Formulations and kits for forming bioadhesive matrices

ABSTRACT

A bioadhesive formulation, comprising gelatin, alginate and a coupling agent, capable of forming a bioadhesive matrix, which is characterized by rapid curing, optimal viscosity, high bonding strength, flexibility, biocompatibility and biodegradability, is disclosed. Further disclosed is such a bioadhesive formulation which further comprises a bioactive agent, and a drug-eluting bioadhesive matrix formed therefrom, the bioadhesive matrix being capable of delivering the bioactive agent to a bodily site. Methods utilizing the bioadhesive formulations and matrices in various biological and medical procedures are also disclosed.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/379,303 filed on Aug. 18, 2014, which is a National Phase of PCTPatent Application No. PCT/IL2013/050139 having International FilingDate of Feb. 14, 2013, which claims the benefit of priority under 35 USC§ 119(e) of U.S. Provisional Patent Application No. 61/599,486 filed onFeb. 16, 2012. The contents of the above applications are allincorporated by reference as if fully set forth herein in theirentirety.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates tobioadhesive matrices, and more particularly, but not exclusively, tobioadhesive matrices, to formulations and kits for forming same and touses thereof for adhering two or more objects, including biologicalobjects such as viable tissues, organs and the likes.

In recent years there has developed an increased interest in replacingor augmenting sutures and staples with bioadhesive matrices (alsoreferred to in the art as bioadhesive compositions). The reasons forthis increased interest include the potential speed with which internalsurgical procedures might be accomplished; the ability of a bondingsubstance to effect complete closure, thus preventing seepage of fluids;the possibility of forming a bond without excessive deformation of thetreated tissue; obviate the need for suture removal; cause less pain tothe patient; its use requires simpler equipment which presents no riskof injury to the practitioner from sharp instruments; it provides lesserscar; and lowers the probability for infections.

Bioadhesives may also be used for sealing air and body fluid leaks,which may occasionally be resistant to conventional suture or staplingtechniques; be used for topical wound closure; repair aorticdissections; and for internal and/or external fixation of certaindevices.

Like any adhesive matrix, bioadhesive matrices are formed upon curing acorresponding formulation. Thus, the formulation is applied onto e.g., abiological object, and when subjected to mixing, curing initiators orother curing initiating conditions, cures so as to afford thebioadhesive matrix.

In order for bioadhesive matrices to be acceptable, they must possess anumber of properties, such as biocompatibility and biodegradability, andoptimal bonding strength and elasticity once cured. Further, thebioadhesive matrices should be designed such that the correspondingformulation exhibits a user-friendly consistency and curing/bondingtime. More specifically, bioadhesive formulations should exhibit optimalinitial viscosity and tack to allow adequate and easy application; nottoo fluid so as not to flow away from the wound edges and not tooviscous so as to interfere with even and proper application, and at thesame time solidify quickly with short curing/gelation time, yet, not tooshort curing/gelation time, so as to allow smooth application to thedesired site. In addition, bioadhesive formulations/matrices shouldexhibit an ability to bond rapidly to living tissue under wet conditionsof bodily fluids; the bioadhesive matrix should form a bridge, typicallya permeable flexible bridge; and the bioadhesive formulation, matrixand/or its metabolic (biodegradation) products should not cause localhistotoxic or carcinogenic effects, while not interfering with thebody's natural healing mechanisms.

Bioadhesives can be used in many surgical procedures including cornealperforations, episiotomy, caesarian cases, cleft lip, skin and bonegrafting, tendon repair, hernia, thyroid surgery, periodontal surgery,gingivectomy, dental implants, oral ulcerations, gastric varices woundsof internal organs such as liver and pancreas, attachment andimmobilization of external and internal medical devices and more.

Fixing fractured hard tissues using an appropriate bioadhesive materialinstead of the traditional nailing and plating methods is alsoconsidered an attractive technique.

The advantages include providing an optimal load transfer from onefracture side to the other, avoiding stress-shielding phenomena and theability to repair small or thin bone fragments. Yet, in spite of theclear medical necessity, there is no hard tissue bioadhesive productavailable for clinical use to date.

Several materials useful as tissue adhesives or tissue sealants arecurrently available.

One type of adhesive that is currently available is a cyanoacrylateadhesive.

Cyanoacrylates, such as 2-octyl cyanoacrylate, known as Dermabond®,create a strong bond to tissue, enables rapid hemostasis and have theability to polymerize in contact with fluids that are present at thebiological surfaces. However, cyanoacrylate adhesives were found to becytotoxic, the viscosity of the pre-cured adhesive formulation is toolow and the cured cyanoacrylate matrix is stiff and non-biodegradable,interfering with normal wound healing. Hence, non-optimal viscosity,high flexural modulus and reports of cancer in animal experimentslimited the use of cyanoacrylates to surface application on oral mucosaand life threatening arteriovenous.

Other known bioadhesive formulations are based ongelatin-resorcinol-formaldehyde, wherein a mixture of gelatin andresorcinol is warmed and crosslinked within tens of seconds by theaddition of formaldehyde. The advantage of bioadhesives formed from suchformulations is adequate bonding strength; however, cytotoxicityovershadows the advantages.

Another type of a currently available bioadhesive which is used as atissue sealant utilizes components derived from bovine and/or humansources. For example, fibrin-based adhesive formulations are typicallyprepared by mixing a solution of fibrinogen and factor XIII with asolution of thrombin and CaCl₂. The two solutions are applied by a twinsyringe equipped with a mixing nozzle, and the reaction is similar towhite fibrin clot in blood clotting. Commercially available examplesinclude Baxter Tisseel® and Ethicon Crosseal™. Advantages offibrin-based bioadhesive matrices include hemostatic effect,biodegradability, good adherence to connective tissue and promotion ofwound healing. Disadvantages include low strength (adhesive andcohesive), low viscosity (hard to apply only to the desired site) andrisk of infection as in use of any human-origin product. In the UnitedStates fibrin adhesives are prepared from the patient's own blood inorder to prevent contamination; however, this process is time consumingand expensive. Other limitations include air leakage in lung surgerythat can reoccur a few days after surgery, possibly due to too-rapidabsorption of the fibrin adhesive bridge.

Other known bioadhesives are protein-based tissue adhesives which arebased on albumin or gelatin. The addition of polyamine, especiallypoly(lysine) or chitosan, or a polycarboxylate, especially citric acidor poly(acrylic acid), to increase the rate of crosslinking was alsodescribed. However, such bioadhesives are typically characterized byinsufficient biocompatibility and strength.

Sung et al. [Journal of Biomedical Materials Research, Volume 46, Issue4, pages 520-530, 15 Sep. 1999] report evaluation of various bioadhesiveformulations including a formulation based on gelatin, alginate andcarbodiimide.

However, the formulations reported by Sung et al., are based on about600 mg/ml gelatin content or higher, which do not afford a workablebioadhesive formulation.

Additional background art include U.S. Patent Application PublicationNos. 20030083286, 20040156794, 20060013873, 20090098176, US20090099149and 20110280952, and U.S. Pat. Nos. 5,284,659 and 5,955,502, and Hsu, S.et al., Biorheology, 2007. 44(1): p. 17-28; Otani, Y. et al.,Biomaterials, 1996. 17(14): p. 1387-1391; Bae, S. K. et al., Journal ofAdhesion Science and Technology, 2002. 16(4): p. 361-372; Mo, X. et al.,Journal of Biomaterials Science, Polymer Edition, 2000. 11(4): p.341-351; McDermott, M. K., et al., Biomacromolecules, 2004. 5(4): p.1270; Mo, X. et al., Journal of Biomedical Materials Research Part A,2010. 94(1): p. 326-332; and Okino, H., et al., Journal of BiomedicalMaterials Research Part A, 2002. 59(2): p. 233-245.

SUMMARY OF THE INVENTION

The present inventors have designed and successfully prepared andpracticed novel bioadhesive formulations capable of forming bioadhesivematrices which are characterized by rapid curing, optimal viscosity,high bonding strength, flexibility, biocompatibility andbiodegradability.

The bioadhesive formulations presented herein comprise gelatin, alginateand a coupling agent, and may further include a bioactive agent, forforming drug-eluting bioadhesive matrices.

The bioadhesive formulations and matrices presented herewith may bebeneficially used in various biological and medical procedures.

Hence, according to an aspect of some embodiments of the presentinvention, there is provided a bioadhesive formulation which comprisesgelatin, alginate, a coupling agent, water and at least one bioactiveagent; the formulation being characterized by a room temperatureviscosity that ranges from 1 Pa-sec to 50 Pa-sec. In some embodiments,the bioadhesive formulation is intended for forming a drug-elutingbioadhesive matrix upon curing, wherein a curing time for forming thematrix ranges from 5 seconds to 30 minutes.

In some embodiments, the bioadhesive formulation is such that the matrixis characterized by at least one of:

a bonding strength of viable biological objects that ranges from 2,000pascal to 60,000 pascal;

a flexural strength at physiological conditions that ranges from 0.5 MPato 200 MPa; and

a biodegradability rate that ranges from 7 days to 6 months.

According to another aspect of some embodiments of the presentinvention, there is provided a bioadhesive formulation which comprisesgelatin, alginate, a coupling agent and water; the formulation beingcharacterized by a room temperature viscosity that ranges from 1 Pa-secto 50 Pa-sec.

In some embodiments, the concentration of gelatin in any of theformulations described herein ranges from 50 mg/ml to 500 mg/ml.

In some embodiments, the concentration of alginate in any of theformulations described herein ranges from 5 mg/ml to 100 mg/ml.

In some embodiments, the concentration of the coupling agent in any ofthe formulations described herein ranges from 1 mg/ml to 50 mg/ml.

In some embodiments, the concentration of gelatin ranges from 200 mg/mlto 300 mg/ml, the concentration of alginate ranges from 20 mg/ml to 40mg/ml and the concentration of the coupling agent ranges from 10 mg/mlto 30 mg/ml.

In some embodiments, a bioadhesive formulation as described hereinfurther includes a crosslinking promoting agent selected from the groupconsisting of an NHS-ester, N-hydroxysuccinimide (NHS), sulfo-NHS, HOBt,HOAt, HBtU, HCtU, HAtU, TBtU, PyBOP, DIC and pentafluorophenol.

In some embodiments, the bioadhesive formulation presented hereinfurther comprises a filler as described herein.

In some embodiments, a bioadhesive formulation as described herein isidentified for use in bonding at least two objects to one another, atleast one of the objects being a biological object.

In some embodiments, the bioadhesive formulation as described herein isprepared ex vivo, in vitro or in situ.

According to another aspect of some embodiments of the presentinvention, there is provided a use of a bioadhesive formulation aspresented herein in the manufacture of a product for use as abioadhesive matrix, and, if the formulation comprises a bioactive agent,for use a drug-eluting bioadhesive matrix.

According to another aspect of some embodiments of the presentinvention, there is provided a use of a formulation as presented hereinin the manufacture of a product for bonding at least two objects to oneanother, at least one of the objects being a biological object.

According to another aspect of some embodiments of the present inventionthere is provided a bioadhesive matrix, formed by curing the formulationpresented herein.

According to another aspect of some embodiments of the present inventionthere is provided a drug-eluting bioadhesive matrix, formed by preparingthe formulation presented herein which includes a bioactive agent, andallowing a curing time to elapse.

According to another aspect of some embodiments of the present inventionthere is provided a kit which includes the bioadhesive formulation aspresented herein.

According to another aspect of some embodiments of the present inventionthere is provided a method of forming a bioadhesive matrix or adrug-eluting bioadhesive matrix, the method being carried out bypreparing the formulation as presented herein and allowing the curing toelapse.

In some embodiments, the method further includes: applying theformulation onto at least one of the objects; and adjoining the objects.

According to another aspect of some embodiments of the present inventionthere is provided a method of bonding at least two objects to oneanother, at least one of the objects being a biological object, themethod being carried out by: preparing the formulation as presentedherein; applying the formulation onto at least one of the objects;adjoining the objects; and allowing a curing time to elapse.

According to another aspect of some embodiments of the present inventionthere is provided a bioadhesive matrix which includes a gelatin and analginate covalently coupled to one another.

In some embodiments, the matrix further comprises at least one bioactiveagent sequestered therein, thereby forming a drug-eluting bioadhesivematrix.

In some embodiments, any of the bioadhesive matrices described herein ischaracterized by at least one of:

a bonding strength of viable biological objects that ranges from 2,000pascal to 60,000 pascal; a flexural strength at physiological conditionsthat ranges from 0.5 MPa to 200 MPa; and a biodegradability rate thatranges from 7 days to 6 months.

In some embodiments, any of the bioadhesive matrices is formed bypreparing the bioadhesive formulation presented herein and allowing acuring time to elapse.

In some embodiments, the preparation of the formulation takes place exvivo, in vitro or in situ.

Unless otherwise defined, all technical and/or scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the invention, exemplarymethods and/or materials are described below. In case of conflict, thepatent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be necessarily limiting.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

Some embodiments of the invention are herein described, by way ofexample only, with reference to the accompanying drawings. With specificreference now to the drawings in detail, it is stressed that theparticulars shown are by way of example and for purposes of illustrativediscussion of embodiments of the invention. In this regard, thedescription taken with the drawings makes apparent to those skilled inthe art how embodiments of the invention may be practiced.

In the drawings:

FIG. 1 presents a comparative bar plot showing the effect of gelatin andalginate concentrations in exemplary bioadhesive formulations, accordingto some embodiments of the present invention, on the bonding strength ofthe resulting bioadhesive matrix, wherein EDC concentration in theformulation is 4 mg/ml, gelatin concentration varies: 100 mg/ml (leftseries), 150 mg/ml (middle series) and 200 mg/ml (right series), andalginate concentration varies: 20 mg/ml (black bars), 40 mg/ml (greybars) and 60 mg/ml (white bars);

FIG. 2 presents a bar plot showing the effect of coupling agent (EDC)concentration in exemplary bioadhesive formulations, according to someembodiments of the present invention, on the bonding strength of theresulting bioadhesive matrix, varying 0, 4, 10, 15 and 20 mg/ml EDC inthe formulation, wherein gelatin is at a concentration of 200 mg/ml andalginate is at a concentration of 40 mg/ml in the formulation;

FIG. 3 presents a scatter plot showing the bonding strength of anexemplary bioadhesive matrix according to some embodiments of thepresent invention, which was afforded from an exemplary bioadhesiveformulation comprising 200 mg/ml gelatin, 40 mg/ml alginate, and 20mg/ml EDC, as a function of time after forming the pre-curingbioadhesive formulation by mixing the gelatin-alginate mixture with thecoupling agent EDC;

FIG. 4 presents a scatter plot showing the water uptake (swelling ratio)as a function of immersion time of exemplary bioadhesive matrices, asmeasured for an exemplary bioadhesive matrix according to someembodiments of the present invention, afforded from an exemplarybioadhesive formulation comprising 200 mg/ml gelatin, 40 mg/ml alginate,and 20 mg/ml EDC;

FIG. 5 presents a bar plot showing the effect of bupivacaine content onthe bonding strength of exemplary bioadhesive matrices, according tosome embodiments of the present invention, afforded from an exemplarybioadhesive formulation comprising 200 mg/ml gelatin, 40 mg/ml alginate,and 20 mg/ml EDC, wherein “A” represents drug-free samples, “B”represents samples loaded with 1% w/v bupivacaine, “C” represents 2% w/vbupivacaine and “D” represents samples loaded with 3% w/v bupivacaine;

FIG. 6 presents a bar plot showing the effect of ibuprofen content onthe bonding of exemplary bioadhesive matrices, according to someembodiments of the present invention, afforded from an exemplarybioadhesive formulation comprising 200 mg/ml gelatin, 40 mg/ml alginate,and 20 mg/ml EDC, wherein “A” represents drug-free samples, “B”represents samples loaded with 1% w/v ibuprofen, “C” represents 2% w/vibuprofen and “D” represents samples loaded with 3% w/v ibuprofen;

FIG. 7 presents a comparative scatter plot, showing the effect ofgelatin concentration on the release profile of bupivacaine fromexemplary bioadhesive drug-eluting matrices according to someembodiments of the present invention (afforded from exemplarybioadhesive formulations comprising 40 mg/ml alginate, 20 mg/ml EDC, 3%w/v bupivacaine and two gelatin concentrations), wherein the resultsobtained for the formulation having 100 mg/ml gelatin are marked byrhombs, and the results of the formulation having 200 mg/ml gelatin aremarked by rectangles;

FIG. 8 presents a comparative scatter plot, showing the effect ofalginate concentration on the release profile of bupivacaine fromexemplary bioadhesive drug-eluting matrices according to someembodiments of the present invention (afforded from exemplarybioadhesive formulations comprising 200 mg/ml gelatin, 20 mg/ml EDC, 3%w/v bupivacaine and three alginate concentrations), wherein the resultsobtained for the formulation having 20 mg/ml alginate are marked byrhombs, the results of the formulation having 40 mg/ml alginate aremarked by rectangles, and the results of the formulation having 60 mg/mlalginate are marked by triangles;

FIG. 9 presents a comparative scatter plot, showing the effect of EDCconcentration on the release profile of bupivacaine from exemplarybioadhesive matrices according to some embodiments of the presentinvention (afforded from exemplary bioadhesive formulations comprising200 mg/ml gelatin, 40 mg/ml alginate, 3% w/v bupivacaine and five EDCconcentrations), wherein the results obtained for the formulation havingno EDC are marked by circles, the results of the formulation having 4mg/ml EDC are marked by X, the results of the formulation having 10mg/ml EDC are marked by triangles, the results of the formulation having15 mg/ml EDC are marked by rhombs, and the results obtained for theformulation having 20 mg/ml EDC are marked by rectangles;

FIG. 10 presents a comparative scatter plot, showing the effect ofbupivacaine concentration on the release profile of bupivacaine fromexemplary bioadhesive drug-eluting matrices according to someembodiments of the present invention (afforded from exemplarybioadhesive formulations comprising 200 mg/ml gelatin, 40 mg/mlalginate, 20 mg/m EDC and three bupivacaine concentrations), wherein theresults obtained for the formulation having 1% w/v bupivacaine aremarked by triangles, the results of the formulation having 2% w/vbupivacaine are marked by rhombs, and the results of the formulationhaving 3% w/v bupivacaine are marked by rectangles;

FIG. 11 presents a comparative scatter plot, showing the effect of EDCconcentration on the release profile of ibuprofen from exemplarybioadhesive drug-eluting matrices according to some embodiments of thepresent invention (afforded from exemplary bioadhesive formulationscomprising 200 mg/ml gelatin, 40 mg/ml alginate, 3% w/v ibuprofen andthree EDC concentrations), wherein the results obtained for theformulation having 10 mg/ml EDC are marked by triangles, the results ofthe formulation having 15 mg/ml EDC are marked by rhombs, and theresults of the formulation having 20 mg/ml EDC are marked by rectangles;

FIG. 12 presents a comparative scatter plot, showing the effect ofibuprofen concentration on the release profile of ibuprofen fromexemplary bioadhesive drug-eluting matrices according to someembodiments of the present invention (afforded from exemplarybioadhesive formulations comprising 200 mg/ml gelatin, 40 mg/mlalginate, 20 mg/m EDC and three ibuprofen concentrations), wherein theresults obtained for the formulation having 1% w/v ibuprofen are markedby triangles, the results of the formulation having 2% w/v ibuprofen aremarked by rhombs, and the results of the formulation having 3% w/vibuprofen are marked by rectangles;

FIG. 13 presents a comparative bar plot, showing the effect of gelatinconcentration on the water uptake of exemplary bioadhesive matricesafforded from exemplary bioadhesive formulations comprising 40 mg/mlalginate and 20 mg/ml EDC, wherein the white bar represents the resultobtained for the bioadhesive formulation containing 100 mg/ml gelatinand the grey bar represents the result obtained for the bioadhesiveformulation containing 200 mg/ml gelatin;

FIG. 14 presents a comparative bar plot, showing the effect of alginateconcentration on the water uptake of exemplary bioadhesive matricesafforded from exemplary bioadhesive formulations comprising 200 mg/mlgelatin and 20 mg/ml EDC, wherein the white bar represents the resultobtained for the bioadhesive formulation containing 20 mg/ml alginateand the grey bar represents the result obtained for the bioadhesiveformulation containing 40 mg/ml alginate;

FIG. 15 presents a comparative bar plot, showing the effect of EDCconcentration on the water uptake of exemplary bioadhesive matricesafforded from exemplary bioadhesive formulations comprising 200 mg/mlgelatin and 40 mg/ml alginate, wherein the white bar represents theresult obtained for the bioadhesive formulation containing 10 mg/ml EDCand the grey bar represents the result obtained for the bioadhesiveformulation containing 20 mg/ml EDC;

FIG. 16 presents a comparative bar plot showing the changes in cellviability of fibroblasts in the presence of medium that was previouslyincubated for 24 hours with the components of exemplary bioadhesiveformulations, according to some embodiments of the present invention,namely gelatin or alginate, while cells kept in the presence of regularmedium served as control, wherein white bars represent results obtainedat time point 0 and the black bars represent the results obtained after24 hours; and

FIGS. 17A-17B present comparative bar plots showing the changes inviability of fibroblasts in the presence of exemplary bioadhesivematrices, according to some embodiments of the present invention,afforded from exemplary bioadhesive formulations comprising 200 mg/mlgelatin and 40 mg/ml alginate (FIG. 17A), or 300 mg/ml gelatin and 30mg/ml alginate (FIG. 17B) and different concentrations of EDC (0, 5, 10,15 and 20 mg/ml), wherein white bars represent results obtained after 24hours and the black bars represent the results obtained after 48 hours;

FIGS. 18A-B present comparative bar-plots, showing the cytotoxic effectof bupivacaine (FIG. 18A) and ibuprofen (FIG. 18B) concentrations insolutions on fibroblast cells based on Alamar-Blue reduction testswhereas significant differences are marked with “-*-”;

FIGS. 19A-19D present ESEM fractographs of bioadhesive matrices,prepared using gelatin (200 mg/ml), alginate (40 mg/ml) and EDC (20mg/ml) without the addition of a drug (FIG. 19A) and loaded with 1% w/vbupivacaine (FIGS. 19B-D), at different magnification power;

FIGS. 20A-20B present ESEM fractographs of bioadhesive matrices,prepared using gelatin (200 mg/ml), alginate (40 mg/ml) and EDC (20mg/ml) loaded with 1% w/v ibuprofen) at different magnification power;

FIGS. 21A-21B present comparative bar plots showing the effect of HA(FIG. 21A) and β-TCP (FIG. 21B) on the bonding strength of exemplarybioadhesive matrices according to some embodiments of the presentinvention, composed of gelatin (200 mg/ml), alginate (40 mg/ml) and EDC(20 mg/ml) to soft tissues, whereas significant differences are markedwith “*”;

FIGS. 22A-22B present comparative bar plots showing the effect of HA(FIG. 22A) and β-TCP (FIG. 22B) on the bonding strength of exemplarybioadhesive matrices according to some embodiments of the presentinvention, composed of gelatin (200 mg/ml), alginate (40 mg/ml) andreduced EDC (10 mg/ml) to soft tissues, whereas significant differencesare marked with “*”;

FIG. 23 presents a bar plot showing the effect of the addition of HA andβ-TCP on the bonding strength of selected bioadhesive formulationscomposed of gelatin (200 mg/ml), alginate (40 mg/ml) and EDC (20 mg/ml)to hard tissue (bone), whereas significant differences are marked with“-*-”;

FIGS. 24A-24F present ESEM fractographs of bioadhesive matrices madefrom bioadhesive formulations, according to embodiments of the presentinvention, comprising gelatin (200 mg/ml), alginate (40 mg/ml) and EDC(20 mg/ml) as well as 0.125% w/v HA (FIG. 24A), 0.5% w/v HA (FIGS. 24Band 24C at different magnification), 0.125% w/v β-TCP (FIG. 24D) and0.5% w/v β-TCP (FIGS. 24E and 24F at different magnification);

FIGS. 25A-25B present bar plots showing the effect of varying the EDCconcentration on the bonding strength of a bioadhesive matrix based on abioadhesive formulation containing 200 mg/ml gelatin and 40 mg/ml LValginate (Ge-200:A1-40, FIG. 25A), and 300 mg/ml gelatin and 30 mg/ml LValginate (Ge-300:Al-30, FIG. 25B), whereas significant differences areindicated by “*”;

FIGS. 26A-26B present bar plots showing the effect of the gelatin'sBloom number on the bonding strength of a bioadhesive matrix resultingfrom a bioadhesive formulation composed of 200 mg/ml gelatin, 40 mg/mlLV alginate and 20 mg/ml EDC (FIG. 26A), and on the viscosity of theircorresponding Ge:Al solutions without EDC (FIG. 26B), whereassignificant differences are indicated by “*”;

FIGS. 27A-27C present bar plots showing the effect of the gelatin andalginate concentrations on the bonding strength of the bioadhesivematrix made from formulations comprising 20 mg/ml EDC, various LValginate concentrations and 200 mg/ml gelatin (FIG. 27A), 300 mg/mlgelatin (FIG. 27B) and 400 mg/ml gelatin (FIG. 27C), whereas significantdifferences are indicated by “*”;

FIGS. 28A-28C present comparative bar plot showing the effect ofalginate's viscosity on the bonding strength of bioadhesive matricesmade from bioadhesive formulations according to embodiments of thepresent invention, having different gelatin concentrations (Bloomnumber=90-110) of 200 mg/ml (FIG. 28A), 300 mg/ml (FIG. 28B) and 400mg/ml gelatin (FIG. 28C), whereas black bars represent LV alginate,white bars represent HV alginate, the alginate concentrations areindicated in the x axis and EDC concentration is 20 mg/ml for all test(significant differences between the bonding strengths of adhesives withsimilar concentrations but different type of alginate are indicated by“*” and significant differences between the bonding strengths of variousHV alginate adhesives are indicated by “v”);

FIG. 29 presents a comparative bar plot showing the effect of thealginate concentration and its viscosity on the viscosity ofgelatin-alginate solutions containing gelatin with a Bloom number of90-110 and a concentration of 200 mg/ml, whereas black bars represent LValginate, white bars represent HV alginate;

FIG. 30 presents a schematic flow-chart representation of a qualitativemodel describing the effects of the bioadhesive formulation components'parameters on the bonding strength, whereas the boxed arrow representsan embodiment where a decrease in a certain parameter results in anincrease in the following one while all other embodiments, an increasein a certain parameter leads to an increase in the following one;

FIGS. 31A-31B present a comparative bar plot showing the combined effectof EDC and NHS on the bonding strength of a gelatin-alginate basedbioadhesive formulation, according to some embodiments of the presentinvention, whereas the gelatin concentration is 200 mg/ml, alginateconcentration is 40 mg/ml, and the numbers on the bars denote thepercent of NHS in the formulation (FIG. 31A), and a bar plot comparingthe bioadhesive matrices exhibiting the highest bonding strength (FIG.31B); and

FIGS. 32A-32B present bar plots showing the effect of the exemplaryantibiotic drug clindamycin on the bonding strength of an exemplarybioadhesive matrix afforded from an exemplary bioadhesive formulation,according to embodiments of the present invention, comprising 200 mg/mlgelatin, 40 mg/ml alginate, and 20 mg/ml EDC, whereas the numbers on thebars denote the percent of clindamycin in the formulation (FIG. 32A),and the effect of the addition of NHS and clindamycin on the bondingstrength of a matrix resulting from a bioadhesive formulation comprising200 mg/ml gelatin, 40 mg/ml alginate, and 10 mg/ml EDC (FIG. 32B).

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates tobioadhesive matrices, and more particularly, but not exclusively, tobioadhesive matrices, to formulations and kits for forming same and touses thereof for adhering two or more objects, including biologicalobjects such as viable tissues, organs and the likes.

The principles and operation of the present invention may be betterunderstood with reference to the figures and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details set forth in the following description or exemplified bythe Examples. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for the purposeof description and should not be regarded as limiting.

While reducing the present invention to practice, the present inventorshave developed and studied highly effective bioadhesive formulationswhich are based on gelatin, alginate and a coupling agent such as EDC,and which can be effectively used, inter alia, for adhering softtissues.

While further developing these bioadhesive formulations, it was shownthat these bioadhesive formulations form bioadhesive matrices which cansequester and subsequently elute effectively a bioactive agent, such asvarious anesthetic and analgesic drugs. The present inventors havepracticed this novel concept of drug-eluting bioadhesive matrices usingbupivacaine and ibuprofen as exemplary bioactive agents. The effect ofthe components of the bioadhesive formulation on the bonding strengthand on the drug release profile of the resulting bioadhesive matriceswas studied, as well as the biocompatibility of these bioadhesivematrices.

While further developing these bioadhesive formulations, it was foundthat the addition of certain fillers, such as hydroxyapatite (HA) andbeta-tricalcium phosphate (β-TCP), affords a highly effectivebioadhesive for hard tissue. While reducing some of the embodiments ofthe present invention to practice, the potential of these fillers toincrease the bonding strength of a bioadhesive matrix to hard tissueshas also been demonstrated; HA and β-TCP were found to improve thebioadhesive matrices, thus affording bioadhesive formulations which canbe used for both soft and hard tissue applications.

As used herein, the term “bioadhesive” refers to a feature of asubstance (e.g., a formulation or a matrix) according to which when thesubstance interfaces a living tissue, it enables to adhere to thisliving tissue an object such as, for example, another living soft and/orhard tissue, the living tissue itself and/or an inanimate object.

As used herein, a bioadhesive formulation thus refers to a formulationwhich is applied to such a living tissue and/or to the object to beadhered to the living tissue, with the aim of adhering the living tissueand the object; and a bioadhesive matrix refers to the substance thatbinds together the living tissue and the object adhered thereto.

According to an aspect of some embodiments of the present invention,there is provided a bioadhesive formulation which comprises:

a) gelatin;

b) alginate;

c) a coupling agent; and

d) water.

It is noted herein and discussed in detailed hereinbelow, that accordingto some embodiments of the present invention, the formulation is definedas a collection of ingredients, which may be found in the form of oneconcoction wherein all ingredients are mixed into the same mixture, orin the form of separate quantified ingredients, for example, separatequantified solvents and dry ingredients.

The bioadhesive formulation, as referred to herein, is a precursor of acorresponding bioadhesive matrix.

In some embodiments, the bioadhesive matrix is formed by curing thebioadhesive formulation, such that the bioadhesive formulation can beregarded as a pre-curing formulation.

Basic Ingredients of the Bioadhesive Formulation:

A bioadhesive formulation, as disclosed herein, contains at least twotypes of polymers (e.g., alginate and gelatin) and a coupling agent.

The bioadhesive formulation is designed to undergo a crosslinkingreaction in the presence of the coupling agent.

Without being bound by any particular theory, it is assumed that thecrosslinking reaction involves crosslinking of gelatin strands withalginate strands, essentially by coupling primary amines in the gelatinto carboxyl groups in the alginate, and/or with one or more othergelatin molecules. When using a fluid formulation comprising gelatin asa major component and alginate as a minor component relative to gelatin,it is assumed that most of the crosslinks form between gelatin andalginate. This network of crosslinked polymers, being substantiallygelatin and alginate, referred to herein as a matrix, is the curedsemi-solid, gel product of the fluid bioadhesive formulation. Hence, thematrix is regarded as a coupled gelatin-alginate matrix afforded bycoupling gelatin and alginate using a coupling agent.

As used herein, and is known in the art, the term “gelatin” describes awater soluble protein that can form gel under certain conditions.Gelatin is typically obtained by heat dissolution at acidic or alkalineand partially hydrolyzing conditions of collagen.

Type A gelatin is obtained by acidic process and has a high density ofamino groups causing a positive charge. Type B gelatin is obtained byalkaline process and has high density of carboxyl groups causingnegative charge. There are different sources for collagen such as animalskin and bone, which afford a variety of gelatin forms with a range ofphysical and chemical properties. Typically, gelatin contains eighteenamino acids that are linked in partially ordered fashion; glycine oralanine is about a third to half of the residues, proline orhydroxyproline are about one fourth and the remaining forth includeacidic or basic amino acid residues. Typically, in order to dissolvegelatin in water it is necessary to reach a temperature of at least 35°C. by heating or stirring and adding hot water. Moderate heatingenhances solubility and severe heating may cause aggregation or partialhydrolysis of gelatin. The viscosity of gelatin varies with type,concentration, time and temperature. Acid processed gelatin has slightlygreater intrinsic viscosity compared to alkali processed gelatin.Gelatin is relatively cheap, it is biocompatible with negligibleimmunologic problems, and it is biodegradable. “Bloom” is a test tomeasure the strength of a gel or gelatin. The test determines the weight(in grams) needed by a probe (normally with a diameter of 0.5 inch) todeflect the surface of the gel 4 mm without breaking it. The result isexpressed in Bloom grades or Bloom number, and it is typically between30 and 300 Bloom. To perform the Bloom test on gelatin, a 6.67% gelatinsolution is kept for 17-18 hours at 10° C. prior to being tested.

In the context of embodiments of the present invention, alternatives togelatin may include non-animal gel sources such as agar-agar (a complexcarbohydrate harvested from seaweed), carrageenan (a complexcarbohydrate harvested from seaweed), pectin (a colloidal carbohydratethat occur in ripe fruit and vegetables), konjaka (a colloidalcarbohydrate extracted from plants of the genus Amorphophallus), guargum (guaran, a type of galactomannan extracted from cluster beans of thegenus Cyamopsis tetragonolobus) and various combinations thereof with orwithout gelatin.

As used herein, and is known in the art, the term “alginate” describesan anionic polysaccharide. Alginate, which is also referred to hereinand in the art as alginic acid, is a block copolymer composed of β-Dmannuronic acid monomers (M blocks) and α-L guluronic acid (G blocks),with different forms of alginate having different ratio of M/G. The term“alginate”, as used herein, encompasses various M/G ratio. M/G ratiovaries according to the species, source and harvest season of thealgae/plant.

In some embodiments, the alginate has an M/G ratio that ranges from 0.3to 4, from 0.7 to 3, or from 1 to 2. In other embodiments, the M/G ratiois 0.7, 0.9, 1, 1.3, 1.5, 1.7, 1.9, 2, 2.3, 2.5, 2.7, 3, 3.5 or 4.

Alginate is known to form a viscous gum by binding water (capable ofabsorbing 200-300 times its own weight in water).

Alginate undergoes reversible gelation in aqueous solution under mildconditions through interactions with divalent cations that bind betweenG-blocks of adjacent alginate chains creating ionic inter chain bridges.Since alginate is generally anionic polymer with carboxyl end, it isknown and used as a good mucoadhesive agent.

Naturally occurring alginate is typically produced in marine brown algae(e.g., Macrocystis pyrifera, Ascophyllum nodosum and Laminaria) and soilbacteria (Pseudomonas and Azotobacter). Synthetically prepared alginatesare also contemplated.

Alginate is relatively cheap, it is biocompatible, evokes no immunologicresponse in mammals, and it is biodegradable.

In the context of some embodiments of the present invention, alginatecan be used in a high-viscosity (HV) form, exhibiting more than 2Pa-sec, or low-viscosity (LV) form, exhibiting 0.1-0.3 Pa-sec. Asdemonstrated in the Examples section hereinbelow, use of the LV/HValginate forms adds another parameter to the fine-tuning andoptimization of the bioadhesive formulation presented herein.

The term “coupling agent”, as used herein, refers to a reagent that cancatalyze or form a bond between two or more functional groupsintra-molecularly, inter-molecularly or both. Coupling agents are widelyused to increase polymeric networks and promote crosslinking betweenpolymeric chains, hence, in the context of some embodiments of thepresent invention, the coupling agent is such that can promotecrosslinking between polymeric chains; or such that can promotecrosslinking between amino functional groups and carboxylic functionalgroups, or between other chemically compatible functional groups ofpolymeric chains; or is such that can promote crosslinking betweengelatin and alginate. In some embodiments of the present invention theterm “coupling agent” may be replaced with the term “crosslinkingagent”. In some embodiments, one of the polymers serves as the couplingagent and acts as a crosslinking polymer.

By “chemically compatible” it is meant that two or more types offunctional groups can react with one another so as to form a bond.

Exemplary functional groups which are typically present in gelatins andalginates include, but are not limited to, amines (mostly primary amines—NH₂), carboxyls (—CO₂H), sulfhydryls and hydroxyls (—SH and —OHrespectively), and carbonyls (—COH aldehydes and —CO— ketones).

Primary amines occur at the N-terminus of polypeptide chains (called thealpha-amine), at the side chain of lysine (Lys, K) residues (theepsilon-amine), as found in gelatin, as well as in various naturallyoccurring polysaccharides and aminoglycosides.

Because of its positive charge at physiologic conditions, primary aminesare usually outward-facing (i.e., found on the outer surface) ofproteins and other macromolecules; thus, they are usually accessible forconjugation.

Carboxyls occur at the C-terminus of polypeptide chain, at the sidechains of aspartic acid (Asp, D) and glutamic acid (Glu, E), as well asin naturally occurring aminoglycosides and polysaccharides such asalginate. Like primary amines, carboxyls are usually on the surface oflarge polymeric compounds such as proteins and polysaccharides.

Sulfhydryls and hydroxyls occur in the side chain of cysteine (Cys, C)and serine, (Ser, S) respectively. Hydroxyls are abundant inpolysaccharides and aminoglycosides.

Carbonyls as ketones or aldehydes can be form in glycoproteins,glycosides and polysaccharides by various oxidizing processes, syntheticand/or natural.

According to some embodiments of the present invention, the couplingagent can be selected according to the type of functional groups and thenature of the crosslinking bond that can be formed therebetween. Forexample, carboxyl coupling directly to an amine can be afforded using acarbodiimide type coupling agent, such as EDC; amines may be coupled tocarboxyls, carbonyls and other reactive functional groups byN-hydroxysuccinimide esters (NHS-esters), imidoester, PFP-ester orhydroxymethyl phosphine; sulfhydryls may be coupled to carboxyls,carbonyls, amines and other reactive functional groups by maleimide,haloacetyl (bromo- or iodo-), pyridyldisulfide and vinyl sulfone;aldehydes as in oxidized carbohydrates, may be coupled to other reactivefunctional groups with hydrazide; and hydroxyl may be coupled tocarboxyls, carbonyls, amines and other reactive functional groups withisocyanate.

Hence, suitable coupling agents that can be used in some embodiments ofthe present invention include, but are not limited to, carbodiimides,NHS-esters, imidoesters, PFP-esters or hydroxymethyl phosphines.

A carbodiimide is a complete crosslinker that facilitates the directcoupling (conjugation) of carboxyls to primary amines. Thus, unlikeother reagents, carbodiimide is a zero-length crosslinker; it does notbecome part of the final crosslink between the coupled molecules.Because peptides, proteins, polysaccharides and aminoglycosides containmultiple carboxyls and amines, direct carbodiimide-mediatedcoupling/crosslinking usually causes random polymerization ofpolypeptides.

EDC, or N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride, isa widely used carbodiimide-type coupling agent and crosslinker whichenables the condensation between carboxyl and amino groups to form amidebonds and the byproduct urea. Once reacted with amine/hydroxylreactants, EDC is not present in the structure of the coupled product;hence its biocompatibility and biodegradability are not an issue in thecontext of the present embodiments. As a gelatin molecule exhibits bothcarboxyl and amino groups, this type of polymer may undergointermolecular crosslinking by EDC.

It is known that EDC and its urea derivative are cytotoxic and inhibitcell growth. This high reactivity towards amino and carboxyl groups inthe living tissues, as well as the release of a urea derivative, areprobably the basis for EDC's cytotoxicity.

Alternatives for carbodiimide-type coupling agent, according to someembodiments of the present invention, include without limitation,glyoxal, formaldehyde, glutaraldehyde, polyglutaraldehyde, dextran,citric acid derivatives, microbial transglutaminase and genipin.

In some embodiments, the coupling agent is used up during the couplingreaction, and produces a urea derivative as a byproduct of the couplingreaction between amine and carboxyl groups. The nature of the ureaderivative is determined by the nature of the coupling agent used.

According to some embodiments of the present invention, various couplingand crosslinking agents may be combined or used as additives in anygiven bioadhesive formulation based on gelatin and alginate and acoupling agent, so as to further promote the crosslinking reaction. In arepresentative example, NHS-esters are added to a carbodiimide-typecoupling agent such as EDC.

The addition of NHS to the crosslinking reaction of EDC affords anNHS-activated carboxylic acid group, which is less susceptible tohydrolysis and prevents rearrangements. On the other hand, at highconcentration NHS can react with the EDC and compete with thecrosslinking reaction, thereby reducing the effective amount of EDC forcrosslinking. Hence, reagents such as NHS are referred to herein acrosslinking promoting agents.

By adding various agents that promote the coupling reaction, and, in thecontext of the present invention, promote the formation of crosslinks inthe forming bioadhesive matrix, it is intended to increase thecrosslinking efficiency and/or reduce the amount of coupling agentneeded to form a matrix the exhibits the desired characteristics, asdiscussed hereinabove. Hence, such agents are referred to herein as“crosslinking promoting agents”. The amount of a crosslinking promotingagent is given as weight/volume per weight/volume percents (w/v/w/v),i.e. relative to the amount of the coupling agent, and according to someembodiments of the present invention, this amount ranges from about 1%to 100%, or from 1% to 200% weight/volume per weight/volume percents.

Representative examples of crosslinking promoting agents include,without limitation, sulfo-NHS, HOBt, HOAt, HBtU, HCtU, HAtU, TBtU,PyBOP, DIC pentafluorophenol and the likes.

As demonstrated in the Examples section that follows, a combination of acrosslinking agent and a crosslinking promoting agent in the bioadhesiveformulations presented herein, affords bioadhesive matrices withimproved bonding strength.

Furthermore, the combination of a crosslinking agent such as EDC and acrosslinking promoting agent such as N-hydroxysuccinimide (NHS), alloweda significant reduction in the EDC content of the bioadhesiveformulation. A reduction of the content of EDC is beneficial due to themedical safety and cytotoxicity implications of using EDC.

In some embodiments, the amount of the crosslinking promoting agent maybe 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40,45, 50, 60, 70, 100, 150, 200%, including any value between 1 and 200%relative to the amount of the coupling agent, or can be even higher. Insome embodiments of the present invention, the amount of thecrosslinking promoting agent is 5, 10, 15, 20, 30 or 40% relative to theamount of the coupling agent, including any value from 5 to 40.

When used in tandem, EDC and NHS afford stronger bonding bioadhesivematrices, even when relatively low concentration of EDC is used. Anexemplary formulation comprises an amount of EDC which is 10 mg/ml andan amount of NHS which is 10% relative to the amount of the EDC.

Fillers for Soft and Hard Tissue Bioadhesive Formulations:

In order to provide bioadhesive formulation/matrix exhibiting mechanicaland chemical properties suitable for use in the context of, for example,hard tissues, bioactive fillers may be added to the formulation. Suchfillers are known to promote bone formation and osseointegration, whichare regarded as highly desired for hard tissue bioadhesiveformulations/matrices.

The term “filler”, as used in the context of some embodiments of thepresent invention, refers to a substance, typically based on amineral-rich source, which is biocompatible, non-toxic and able toreinforce the matrix mechanically, and in some embodiments, capable ofpromoting bone formation and osseointegration. In some embodiments, a“filler” substance is present in the bioadhesive formulation presentedherein in the form of a fine powder of an aqueous-insoluble substance,namely having low solubility in aqueous media.

Some fillers, according to some embodiments of the present invention,are also bioresorbable, at least to some significant extent, as can beassessed by their physiologic resorption time. It has been shown thatsome bioresorbable filler substances act as more than simple spacefillers that prevent soft tissue ingrowth. Such filler substances, onceundergone physiologic resorption, may leave a consistent latticework ofminerals, which were found to be stable in the long term and to act asan osteoconductive trellis for new bone formation. It was also found topromote the formation of blood vessels, which is essential for boneformation.

As used herein, the term “resorbable” refers to a substance which canundergo resorption, as this term is defined hereinbelow. When theresorption process takes place in a biologic system, the substance isreferred to as “bioresorbable”.

The term “resorption”, as used herein, describes a loss of a substancethrough chemical, biological and/or physiologic processes. Typically,this term is used herein and in the art to describe such a process whichinvolves decomposition of a substance by, e.g., chemical or physicalbreak-down, such as dissolution, hydrolysis and/or phagocytosis, whichmay be followed by absorption and/or excretion of the breakdown productsby the body via, for example, metabolism. The term resorption istherefore often referred to herein and in the art as “bioresorption”.Accordingly, the phrase “resorption period”, as used herein, refers tothe time period of the resorption process.

Non-limiting examples of fillers, according to some embodiments of thepresent invention, include the following materials listed (some withtheir approximate physiologic resorption time in parentheses): calciumsulfate of all hydration forms, tricalcium phosphate (Ca₃(PO₄)₂) ofvarious crystal form, beta-tricalcium phosphate (β-TCP) (4-12 months),micro-porous hydroxylapatite (HA) particulate (18-36 months),bovine-derived hydroxylapatite with synthetic peptide (18-36 months),calcified algae (6-18 months), synthetic particulate glass ceramic(bioactive glass, 18-24 months), autogenic bone shavings (3-7 months),allogeneic cancellous bone (6-15 months), irradiated cancellousallogeneic bone (4-12 months), inorganic bovine bone (15-30 months),porous anorganic crystal (4-10 months), porous coralline hydroxylapatite(5-7 years) and composite of micro-porous Bioplant HTR polymer coatedwith calcium hydroxide (10-15 years).

It is noted herein that a filler, according to embodiments of thepresent invention, may be in a form of a fine or coarse powder,depending on the application and intended use, namely its particles mayexhibit an average particle size that ranges from about 1 μm in diameterto about 2 mm. In some embodiments, the average particle size of thefiller ranges from 2-30, 5-20, 15-40. 20-50, 50-60 or 50-100 μm indiameter.

As demonstrated in the Examples section that follows, two types ofexemplary bioactive ceramic fillers, hydroxyapatite (HA) andbeta-tricalcium phosphate (β-TCP), were added to the bioadhesiveformulation presented herein in order to afford a bioadhesiveformulation/matrix which exhibits improved bonding strength, while usinglower amounts of a crosslinking agent (e.g., EDC) relative tocorresponding filler-free formulations, and further found to be highlysuitable for hard tissue, such as bones. These two exemplary fillersthere are characterized by good biocompatibility and lack of toxicity,but and as capable of promoting bone formation and osseointegration.

The effect of adding fillers such as HA and β-TCP into bioadhesiveformulation, according to some embodiments of the present invention, hasbeen studied in terms of the microstructure of the resulting bioadhesivematrix and on the resulting bonding strength to both soft and hardtissues ex vivo. It was found that due to their unique interaction withthe aqueous media of the formulation and their low solubility therein,the fillers form fibers and aggregates that presumably act as mechanicalreinforcement of the matrix.

As demonstrated in the Examples section that follows, fillers such as HAand β-TCP can be used in the bioadhesive formulations presented hereinas powders in amounts such as 0.025, 0.05, 0.075, 0.1, 0.125, 0.15, 0.2,0.225, 0.25, 0.275, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 1, 1.5, 2, 3percents weight per volume (% w/v) of the bioadhesive formulation orhigher. According to some embodiments, the use of fillers may allowusing EDC at low concentrations relative to corresponding filler-freebioadhesive formulations. As can be seen in the experimental resultspresented hereinbelow, bioadhesive matrices containing concentrations of0.5% and 0.125% w/v HA or β-TCP respectively according to some exemplaryembodiments of the present invention, were found to have bondingstrength higher than their filler-free counterparts.

Various additives may be added to the formulation in order to modify itspre-curing characteristics, such as for example, viscosity modifiers forimproved application and spread confinement, penetration enhancers, andcolorants or fluorescent agents for allowing tracking during applicationand follow-up; Various additives may be added to the formulation inorder to modify its post-curing characteristics, namely additives thataffect the characteristics of the resulting matrix, such as for example,additional coupling/crosslinking agents, calcium ions and ions of otherearth-metals, which act as gelling agents by virtue of beingcrosslinkers for various alginate species, plasticizes, hardeners,softeners and other agents for modifying the flexural modulus of thematrix, and additives that affect the release rate, penetration andabsorption of the bioactive agent, when present, as discussed in moredetail hereinbelow.

It is noted herein that like the filler, some of the additives, as wellas some of the bioactive agents described herein, may be added to theformulation in the form of a dry powder. This may be for the sake ofsimplicity of use, the wish to maintain decrease or augment certainviscosity value of the bioadhesive formulation, or simply because theadditive of bioactive agent is not soluble in aqueous media.

Characteristics of the Bioadhesive Formulation:

As discussed hereinabove, a design of an effective bioadhesiveformulation should be made while considering several requirements, whichinclude, for example, workability and efficiency, both translated alsointo safety.

Under the workability and safety considerations, an effectivebioadhesive formulation should exhibit a viscosity at room temperaturewhich allows the practitioner to apply and use the formulation under,e.g., clinic conditions. For example, a formulation which is too viscousand thus not spreadable would be difficult to be applied on tissues anda formulation which is not viscous enough and is thus too fluid would berunny and may be accompanied by undesirable leakage, insufficientadhesion and overall, in enhanced adverse side effects and reducedsafety.

Further under the workability and safety consideration, an effectivebioadhesive formulation should exhibit a curing time, as defined herein,which allows completing an operation utilizing the formulation in arelatively short time, so as to avoid prolonged operations which mayresult in enhanced adverse side effects and reduced safety, yet, on theother hand, be sufficiently long to allow accurate positioning andoptional re-positioning if required. In some embodiments instantaneousbonding of objects may be undesired, for example in cases where theobjects are not easily positioned optimally and a re-positioning stepmay be required; hence, exceedingly short curing time may be impracticalin some embodiments of the present invention. In such cases it isdesirable that the bioadhesive formulation allows a period of time forre-positioning (separation and re-adjoining) of the objects to be bondedto one-another. This range of time (window) is referred to as theworkable time of the formulation, as discussed further hereinbelow.

It is noted herein that according to some embodiments of the presentinvention, the formulation can be kept in a number of separated parts,or solutions, at least until all these parts are mixed into a singleconcoction comprising all ingredients in conjunction or concomitant toits application and use. A detailed discussion regarding multi-partformulations, means of storage, preparation and application of thebioadhesive formulation is presented hereinbelow.

When referring to a viscosity of a multi-part bioadhesive formulation,it pertains essentially to the more viscous part(s) of the formulation,namely the part which contains gelatin, alginate or a mixture of gelatinand alginate with or without a bioactive agent or other additives. Thisviscous part will essentially maintain similar viscosity as long as thechemical composition thereof is maintained, namely the concentration ofthe viscosity-conferring polymers (or water content), temperature andmolecular structure.

Alternatively, a reference to viscosity pertains to a formulationcomprising all ingredients shortly subsequent to their mixing. While thedissolved polymers contribute the most to the high viscosity, theaddition of a coupling agent will change the chemical composition of thepolymers by effecting crosslinking which irreversibly alters theviscosity essentially regardless of temperature and water content. Frompractical reasons, when referring herein to viscosity, it is referred toa formulation part containing gelatin and alginate, while the partcontaining the coupling agent is considered less viscous and minor involume relative to the viscous part. Measuring the viscosity of thebioadhesive formulation using standard and common practices andequipment may be impractical due to the relatively short curing time.Gelatin and alginate are the predominant contributors to viscosity,while all other constituents and additives are minor viscositymodifiers. The outstanding constituents are the crosslinking agent andthe crosslinking promoting agent, which have a direct effect on theviscosity of the formulation, as these agents are responsible forhardening the formulation to a solid bioadhesive matrix. Hence, whenmeasuring the viscosity of the formulation one can measure the viscosityof the formulation prior to the addition of the crosslinking agentand/or the crosslinking promoting agent. It is safely assumed that theviscosity of the formulation lacking the hardening agents is essentiallythe same as the viscosity of the complete formulation.

As discussed hereinbelow, the bioadhesive formulation is characterizedby a “workable time”, referring to the time period between the timepoint where all ingredients are mixed together, to the time point wherethe formulation is too viscous to work-up. Hence, the reported viscosityof a bioadhesive formulation, according to some embodiments of thepresent invention, is the effective viscosity during the workable time.

Dynamic viscosity is quantified by various units, depending on themeasuring method and other factors. In the context of the presentembodiments, dynamic viscosity is referred to in units of Newton secondper square meter (N s m⁻² or Pa-sec), wherein 1 Pa-sec is equivalent to1 kilogram per meter second (kg m⁻¹ s⁻¹) and equivalent to 10 poise (P).For example, water at 20° C. are said to have dynamic viscosity of 1mPa-sec (0.001 Pa-sec), blood at 37° C. is characterized by a viscosityof 3-4 mPa-sec, and honey at 20° C. by 10 Pa-sec.

In the context of the present invention, when referring to the viscosityof the bioadhesive formulation, the actual measured results of theformulation lacking the crosslinking agent and/or the crosslinkingpromoting agent are taken and regarded as that of the completebioadhesive formulation.

Thus, according to some embodiments of the present invention, theformulations presented herein are characterized by at least one of:

a room temperature viscosity that ranges from 1 Pa-sec to 50 Pa-sec(referring to either the gelatin-alginate solution, before adding thecoupling agent solution, or to the final bioadhesive formulationcontaining all components just after being mixed together); and

a curing time under physiological conditions that ranges from 5 secondsto 30 minutes.

While the criteria for dynamic viscosity is given in Pa-sec units andits values are derived from particular viscosity measurements at a givenambient temperature, it is noted herein that dynamic viscosity can beexpressed by other units, and measured by various methods andtechniques, all of which can be used to characterize any givenbioadhesive formulation or part thereof. For example, while it issimpler to measure dynamic viscosity at room temperature, it may also beuseful to report and consider also the dynamic viscosity of bioadhesiveformulations at a temperature higher than the working temperature, sinceit is more efficient and practical to mix and prepare the formulation athigher temperatures, such as 50° C. Alternatively, it may be moreinformative to report and consider dynamic viscosity at a temperaturelower that the standard room temperature since, for example, mostoperating rooms are kept at a standard temperature lower than roomtemperature; as well as reporting and considering the viscosity of abioadhesive formulation at or near body temperature, where theformulations is intended to be applied and used.

Hence, according to some embodiments of the present invention, thedynamic viscosity of the bioadhesive formulation, or the dynamicviscosity of the formulation part containing gelatin and alginate,ranges from 1 Pa-sec to 50 Pa-sec at 20° C., and/or 0.5 Pa-sec to 25Pa-sec at 37° C.

According to some embodiments of the present invention, the roomtemperature dynamic viscosity of the bioadhesive formulation, or thedynamic viscosity of the formulation part containing gelatin andalginate, ranges from 1 Pa-sec to 5 Pa-sec, from 5 Pa-sec to 10 Pa-sec,from 10 Pa-sec to 15 Pa-sec, from 15 Pa-sec to 20 Pa-sec, from 20 Pa-secto 25 Pa-sec, from 25 Pa-sec to 30 Pa-sec, from 30 Pa-sec to 35 Pa-sec,from 35 Pa-sec to 40 Pa-sec, from 40 Pa-sec to 45 Pa-sec, or from 45Pa-sec to 50 Pa-sec.

As used herein, the phrase “curing time” describes a time period duringwhich the bioadhesive formulation forms a bioadhesive matrix, asdescribed herein.

It is noted herein that while the bioadhesive formulation begins to cureupon contacting the coupling agent with one or both gelatin and/oralginate, this coupling and crosslinking reaction is not instantaneousacross the entire bulk of the mass of the formulation. Accordingly, theterm “curing time” is defined such that it encompasses the entireprocess of matrix formation at all its stages, including the “workabletime” and the “bonding time”. The “workable time” is the time-windowbetween the moment of mixing all the ingredients of the formulationtogether, to the moment at which the formulation's viscosity is toohigh, presumably due to its curing process, to allow working with theformulation, namely applying, spreading, positioning and re-positioning,as discussed hereinabove. In the context of the viscosity characteristicof the bioadhesive formulation presented hereinabove, the viscosity isrelevant during the workable time, until crosslinking prevails and turnsthe formulation too viscous. The “bonding time” is defined as the timewhich elapses from the moment the formulation is applied on the object,to the moment at which the objects which are being bonded one to theother, are considered bonded at sufficient strength, so as to allow, forexample, release of any fastening/tightening means (if used) and/or thecontinuation or completion of the procedure. The workable time and thebonding time may overlap to some extent, may continue one anotherrespectively or may be discontinued, depending on the formulation, modeof its use as a single or multi-part formulation, the conditions of useand the objects' type and the bonding area.

According to some embodiments of the present invention, the workabletime of the bioadhesive formulation presented herein, containing allingredients mixed together, spans from 5 seconds to 30 minutes.Depending on the required and intended use of the formulation, it can bedesigned to exhibit various workable times which may span at least 30seconds, at least 60 seconds, at least 120 seconds, at least 300seconds, at least 600 seconds, at least 900 seconds, or at least 1800seconds (30 minutes).

According to some embodiments of the present invention, the curing timeof the bioadhesive formulation presented herein ranges from 5 seconds to30 minutes.

Depending on the required and desired performance of the formulation, itcan be designed to exhibit various curing times which may range from 5to 30 seconds, from 5 to 30 seconds, from 5 to 60 seconds, from 5 to 120seconds, from 10 to 300 seconds, from 30 to 300 seconds, and from 60 to1800 seconds (30 minutes). Alternatively, in cases where the adhesionprocess is not restricted in time and other parameters such as strengthand flexibility are more consequential, such as for example in topical(external) adhesion of a device to a patient's skin, the curing time maybe longer than the aforementioned values, and can range from a fewseconds to more than 30 minutes, and be, for example, 40 minutes, 50minutes, 60 minutes, and even 120 minutes, including any intermediatevalue from 30 minutes and 120 minutes.

The Resulting Bioadhesive Matrix:

The bioadhesive matrix is a result of the curing process which takesplace between some of the ingredients of the bioadhesive formulation,and hence the matrix comprises the gelatin and alginate, coupled to oneanother, as discussed herein, and optionally other constituents of theformulation which became associated with the matrix (e.g., entrappedtherein).

As discussed hereinabove, the bioadhesive formulation as describedherein is designed to form a corresponding bioadhesive matrix, and ishence designed such that the bioadhesive matrix exhibits a desiredperformance. According to some embodiments of the present invention, thebioadhesive formulation as described herein is designed such that uponits curing, it forms a bioadhesive matrix that is characterized by ahigh bonding strength of viable biological objects as defined herein, anoptimal flexural modulus under physiological conditions, and an optimalbiodegradability rate.

As used herein, the expression “optimal” relates to the performance ofthe formulation and/or corresponding matrix at a desired application. Itis noted in this regard that different applications may requiredifferent parameters for an optimal performance, which may typicallydepend on the type of objects to be bonded, the dimensions of theobjects to be bonded, the bonding area, the conditions the nature of theprocedure calling for adhesion and other object- and procedure-dependentparameters.

While bonding strength of various viable tissues and other live orinanimate objects is a highly desired characteristic of a bioadhesiveformulation/matrix, it is a non-trivial ability of the bioadhesiveformulations presented herein to bond various objects, such as viableand non-viable tissues, bone, skin, metal, plastic and other natural andsynthetic polymeric substances, under physiological conditions ofmucus/plasma/blood wet environments.

The binding strength can be experimentally determined from the slope ofa stress-strain curve created during tensile tests conducted on a sampleof bonded objects, and expressed in units of force per unit area (Newtonper square meter (N/m²) or dynes per square centimeter), namely Pascals(Pa), megaPascals (MPa) or gigaPascals (GPa).

The phrase “bonding strength” as used herein describes the maximumamount of tensile stress that a pair of bonded objects of givenmaterials can be subjected to before they break apart.

According to some embodiments of the present invention, the bioadhesivematrix afforded from the bioadhesive formulation presented herein ischaracterized by a maximal bonding strength of viable biological objectsthat ranges from about 2,000 pascal (2 MPa) to about 60,000 pascal (60MPa) at peak bonding. According to some embodiments, the maximal bondingstrength of viable biological objects, exhibited by the bioadhesivematrix presented herein ranges from 2 MPa to 10 MPa, from 5 MPa to 20MPa, from 15 MPa to 30 MPa, from 20 MPa to 40 MPa, from 30 MPa to 50MPa, or from 40 MPa to 60 MPa.

Another desired characteristic of the bioadhesive matrix, according tosome embodiments of the present invention, is the extent of its capacityto bend and flex under stress and pressure without breaking or detachingfrom the objects it bonds, while being under physiological conditions,especially wet and swelled by absorbing water from the environment.

Depending on the intended use and conditions, the bioadhesive matrix isexpected to perform (maintain its adhesive role in physiologicalconditions) over time and under intermittent or continuous motion,stress, deformation, bending, stretching, pressure and tear. Accordingto some embodiments of the present invention, the matrix ischaracterized by a flexural strength (modulus) under physiologicalconditions that ranges from 0.5 MPa to 200 MPa. Alternatively, theflexural modulus under physiological conditions, exhibited by thebioadhesive matrix presented herein, ranges from 0.5 MPa to 5 MPa, from1 MPa to 10 MPa, from 5 MPa to 20 MPa, from 10 MPa to 15 MPa, from 15MPa to 20 MPa, from 20 MPa to 30 MPa, from 30 MPa to 50 MPa, from 50 MPato 100 MPa, from 75 MPa to 150 MPa, from 100 MPa to 150 MPa, or from 155MPa to 200 MPa.

According to some embodiments of the present invention, the bioadhesivematrix presented herein is biodegradable.

In order to be used effectively in various internal or external medicalprocedures and particularly in internal surgical procedures, thebioadhesive matrix presented herein further exhibits an optimalbiodegradation rate, allowing it to bond the objects for a sufficientlength of time so as to exhibit its intended use before disintegrating.

The term “biodegradable” and any adjective, conjugation and declinationthereof as used herein, refers to a characteristic of a material toundergo chemical and/or physical transformation from a detectable solid,semi-solid, gel, mucus or otherwise a localized form, to a delocalizedand/or undetectable form such as any soluble, washable, volatile,absorbable and/or resorbable breakdown products or metabolites thereof.A biodegradable material undergoes such transformation at physiologicalconditions due to the action of chemical, biological and/or physicalfactors, such as, for example, innate chemical bond lability, enzymaticbreakdown processes, melting, dissolution and any combination thereof.

Depending on the chemical and physical characteristics of thebioadhesive matrix and the location of its application, and the intendeduse thereof, the process of biodegradation of the matrix can span daysto weeks to months. The phrase “biodegradability rate” is defined hereinas the period of time between application of a bioadhesive formulationto the time by which the resulting bioadhesive matrix is no longerpresent as a bioadhesive matrix. By being “no longer present” it ismeant that substance(s) that can be attributed to the original matrixcan no longer be detected at the site of application of the bioadhesiveformulation at a substantial level, or that traces thereof which maystill be detected in the original site beyond normal levels can nolonger bond tissue or linger at that site.

In general, the bonding strength of the bioadhesive matrix, formed fromthe bioadhesive formulation presented herein, will start to degrade tosome extent under physiological conditions. This degradation in strengthis caused by the breakdown of the matrix, which is effected by acombination of factors, including chemical processes (swelling,dissolution and spontaneous chemical degradation), biological processes(enzymatically driven reactions, formation of new un-bonded cells andother tissue components and death of bonded cells and other tissuecomponents), mechanical processes (stress, strain and tear) and thelikes. For the sake of simplicity, the collection of factors andprocesses that degrade the bonding strength of the bioadhesive matricespresented herein are encompassed and unified under the phrase“biodegradability rate”.

According to some embodiments of the present invention, the intended useof the bioadhesive formulation is to form a matrix that can holdbiological objects attached to one-another for a time period long enoughfor the object to splice, fuse or heal. The period of time depends onthe objects and on the medical procedure being performed.

For example, the formulation may be used to form a matrix that adjoinstwo edges of an incision strong and long enough to allow the incision torepair itself and heal; the incision may be in an internal site in thebody or on the surface (skin and muscle). In another example, one of theobjects is a patch of skin and the other object is an inanimate medicaldevice, in which case the bioadhesive matrix is intended to hold thedevice affixed to the skin until it fulfills its purpose or until thematrix is replaced.

The biodegradability of the bioadhesive matrix can be manipulated by acombination of factors, starting at the composition, namely relativeconcentration of the polymers and the crosslinking density, additivesthat can alter the molecular structure of the matrix (more and variedcrosslinks), biodegradation accelerators/enhancers and biodegradationinhibitors/suppressors. Another factor that can be used to manipulatedegradability is the macroscopic shape and structure of the matrix,namely its surface area, accessibility to the surrounding medium, sizeof the treated area and the likes.

Hence, according to some embodiments of the present invention, thebiodegradability rate of the bioadhesive matrix presented herein rangesfrom about 7 days to about 6 months. In some cases the degradabilityperiod can be made shorter and range from 1 week to 1 month, includingany time period in between, such as from 10 days to 3 weeks. In othercases, the degradability period can be made longer, e.g. 1-6 months, andrange from 1 month to 2 months, from 2 month to 3 months or range from 2months to 6 months.

Another parameter that can be used to determine the time factor involvedin the bonding strength of any given bioadhesive matrix, including thematrix formed from the presently claimed bioadhesive formulation, is thehalf time of bonding strength retention, namely the period of time bywhich the maximal bonding strength reaches half its value, T1/2.

According to some embodiments of the present invention, T1/2 ranges fromabout 1 day to about 5 months and any value therebetween. For example,for short-adhesion period applications, T1/2 ranges from about 1 week toabout two weeks, or from about 10 days to about 1 month. For longeradhesion periods, T1/2 ranges from about 1 month to about 2 months, orfrom about 2 months to about 3 months, or from about 3 months to about 4months, or from about 3 months to about 5 months.

It should be noted herein that while a minimal bonding time and anoptimal biodegradability rate are discussed, the bioadhesive matrixpresented herein can be removed before it is biodegraded, and itsbonding time can be shortened intentionally by mechanical and chemicalmeans.

Concentrations of Basic Ingredients:

The present inventors have uncovered that at least some of theabove-described characteristics of the bioadhesive formulation and thematrix formed therefrom can be manipulated by the relativeconcentrations of each of the ingredients of the bioadhesive formulationshould be optimized.

According to some embodiments of the present invention, a bioadhesiveformulation as described herein comprises gelatin, alginate and acoupling agent, as described herein, each being at a concentration thatwould impart the formulation with the herein-described characteristicsand/or being at relative content ratio that would impart the formulationwith the herein-described characteristics, as presented hereinbelow.

It is noted herein that for the bioadhesive formulation to be useful andeffective, the concentration of its polymers is selected to afford aworkable consistency (primarily in terms of viscosity). Therefore thehigh limit of the range of polymer concentration of workableformulations, and particularly that of gelatin, cannot exceed a certainvalue. Exceeding one or more of these maximal range values may result inan impracticable formulation.

According to some embodiments of the present invention, theconcentration of gelatin in the formulation is 500 mg/ml or lower.

According to some embodiments of the present invention, theconcentration of gelatin in the formulation ranges from 50 mg/ml to 500mg/ml. In some embodiments, the gelatin content in the bioadhesiveformulation ranges from 100 mg/ml to 500 mg/ml, from 100 mg/ml to 400mg/ml, from 100 mg/ml to 300 mg/ml, from 100 mg/ml to 200 mg/ml, or from50 mg/ml to 400 mg/ml, from 50 mg/ml to 300 mg/ml, from 50 mg/ml to 200mg/ml or from 50 mg/ml to 100 mg/ml, including any value between theabove-indicated values.

According to some embodiments of the present invention, theconcentration of alginate in the formulation ranges from 5 mg/ml to 100mg/ml. In some embodiments, the alginate content in the bioadhesiveformulation ranges from 10 mg/ml to 100 mg/ml, from 10 mg/ml to 90mg/ml, from 10 mg/ml to 80 mg/ml, from 10 mg/ml to 70 mg/ml, from 10mg/ml to 60 mg/ml, from 10 mg/ml to 50 mg/ml, from 10 mg/ml to 40 mg/ml,or from 5 mg/ml to 90 mg/ml, from 5 mg/ml to 80 mg/ml, from 5 mg/ml to70 mg/ml, from 5 mg/ml to 60 mg/ml, from 5 mg/ml to 50 mg/ml, from 5mg/ml to 40 mg/ml, from 5 mg/ml to 30 mg/ml, from 5 mg/ml to 20 mg/ml,or from 5 mg/ml to 10 mg/ml, including any value between theabove-indicated values.

According to some embodiments of the present invention, theconcentration of the coupling agent in the formulation ranges from 1mg/ml to 50 mg/ml, from 1 mg/ml to 40 mg/ml, from 1 mg/ml to 30 mg/ml,from 1 mg/ml to 20 mg/ml, or from 1 mg/ml to 10 mg/ml, including anyvalue between the above-indicated values.

As discussed hereinabove, since some coupling agents are known ascytotoxic or as generating cytotoxic moieties, it is desired to userelatively low amount of the coupling agent within a bioadhesiveformulation. On the other hand, reducing the amount of a coupling agentin the formulation may result in reducing the bonding strength of thebioadhesive matrix formed from the formulation. The present inventorshave demonstrated that formulations containing 20-40 mg/ml or even loweramounts of a coupling agent can be used to provide bioadhesive matricesin which the bonding strength is not compromised substantially.

A few exemplary bioadhesive formulations are presented below, eachcomprising gelatin, alginate and a coupling agent at the indicatedconcentrations, given as percent by weight per volume of the totalvolume of the formulation:

Exemplary Formulation I:

100 mg/ml gelatin; 20 mg/ml alginate and 4 mg/ml EDC as acarbodiimide-type coupling agent;

Exemplary Formulation II:

100 mg/ml gelatin; 40 mg/ml alginate and 4 mg/ml EDC;

Exemplary Formulation III:

100 mg/ml gelatin; 60 mg/ml alginate and 4 mg/ml EDC;

Exemplary Formulation IV:

100 mg/ml gelatin; 40 mg/ml alginate and 10 mg/ml EDC;

Exemplary Formulation V:

150 mg/ml gelatin; 40 mg/ml alginate and 10 mg/ml EDC;

Exemplary Formulation VI:

200 mg/ml gelatin; 40 mg/ml alginate and 10 mg/ml EDC;

Exemplary Formulation VII:

200 mg/ml gelatin; 40 mg/ml alginate and 5 mg/ml EDC;

Exemplary Formulation VIII:

200 mg/ml gelatin; 70 mg/ml alginate and 35 mg/ml EDC;

Exemplary Formulation IX:

250 mg/ml gelatin; 40 mg/ml alginate and 25 mg/ml EDC; or

Exemplary Formulation X:

200 mg/ml gelatin; 40 mg/ml alginate and 20 mg/ml EDC; or

Exemplary Formulation XI:

500 mg/ml gelatin; 10 mg/ml alginate and 25 mg/ml EDC.

It is noted herein that, in some embodiments of the present invention,the concentration of the coupling agent may be lowered by the presenceof an additive, without compromising the formulation's performance.

For example, a bioadhesive formulation comprises gelatin, alginate, acoupling agent and a crosslinking promoting agent in the followingamounts, given in weight per volume of the total volume of theformulation:

Exemplary Formulation XII:

200 mg/ml gelatin; 40 mg/ml alginate, 10 mg/ml EDC and 2 mg/mlNHS-ester-type coupling agent (20% relative to EDC concentration);

Exemplary Formulation XIII:

200 mg/ml gelatin; 40 mg/ml alginate, 10 mg/ml EDC and 4 mg/ml NHS-ester(40% relative to EDC concentration);

Exemplary Formulation XIV:

200 mg/ml gelatin; 40 mg/ml alginate, 15 mg/ml EDC and 1.5 mg/mlNHS-ester (10% relative to EDC concentration);

Exemplary Formulation XV:

200 mg/ml gelatin; 40 mg/ml alginate, 15 mg/ml EDC and 3 mg/ml NHS-ester(20% relative to EDC concentration);

Exemplary Formulation XVI:

250 mg/ml gelatin; 30 mg/ml alginate, 20 mg/ml EDC and 4 mg/ml NHS-ester(20% relative to EDC concentration);

Exemplary Formulation XVII:

500 mg/ml gelatin; 10 mg/ml alginate, 20 mg/ml EDC and 4 mg/ml NHS-ester(20% relative to EDC concentration); or

Exemplary Formulation XVIII:

300 mg/ml gelatin; 30 mg/ml alginate, 10 mg/ml EDC and 1 mg/ml NHS-ester(10% relative to EDC concentration).

It is noted herein that, in some embodiments of the present invention,the formulation may include a filler, without compromising theformulation's performance and even improving it and rendering it moresuitable for use in a wider range of applications, such as hard tissueadhesion (bone adhesion etc.).

For example, a bioadhesive formulation comprises gelatin, alginate, acoupling agent, an optional crosslinking promoting agent and a filler inthe following amounts:

Exemplary Formulation XIX:

200 mg/ml gelatin; 40 mg/ml alginate, 20 mg/ml EDC and 0.25% w/vhydroxylapetite;

Exemplary Formulation XX:

200 mg/ml gelatin; 40 mg/ml alginate, 20 mg/ml EDC and 0.5% w/v β-TCP;

Exemplary Formulation XXI:

200 mg/ml gelatin; 40 mg/ml alginate, 10 mg/ml EDC and 0.5% w/vhydroxylapetite;

Exemplary Formulation XXII:

200 mg/ml gelatin; 40 mg/ml alginate, 10 mg/ml EDC and 0.125% w/v β-TCP;

Exemplary Formulation XXIII:

200 mg/ml gelatin; 40 mg/ml alginate, 20 mg/ml EDC, 2 mg/ml NHS-ester(10% relative to EDC concentration) and 0.25% w/v hydroxylapetite;

Exemplary Formulation XXIV:

200 mg/ml gelatin; 40 mg/ml alginate, 20 mg/ml EDC, 4 mg/ml NHS-ester(20% relative to EDC concentration) and 0.5% w/v β-TCP;

Exemplary Formulation XXV:

300 mg/ml gelatin; 30 mg/ml alginate, 10 mg/ml EDC, 1 mg/ml NHS-ester(10% relative to EDC concentration) and 0.5% w/v hydroxylapetite; or

Exemplary Formulation XXVI:

300 mg/ml gelatin; 30 mg/ml alginate, 10 mg/ml EDC, 1 mg/ml NHS-ester(10% relative to EDC concentration) and 0.125% w/v β-TCP.

Thus, in some embodiments, a bioadhesive formulation as describedherein, comprises alginate, gelatin, water, and a coupling agent, asdescribed herein, and further comprises a cross-linking promoting agentand/or a filler, as described herein, wherein the concentration of thecoupling agent is 20 mg/ml or lower (e.g., 15 mg/ml, or 10 mg/ml, orlower).

Any of the aforementioned exemplary formulation may further include oneor more bioactive agents as described herein.

It is noted herein that other combinations of component concentrationsare contemplated, some of which have been demonstrated in the Examplessection that follows.

Drug-Eluting Bioadhesive Formulations:

According to some embodiments of the present invention, a bioadhesiveformulation as described herein further comprises one or more bioactiveagent(s). In some embodiments, such a formulation is designed to afforda drug-eluting bioadhesive matrix upon curing. In other words,bioadhesive formulations which contain a bioactive agent, cure to form adrug-eluting bioadhesive matrix in which the bioactive agent isincorporated. In some embodiments, such drug-eluting bioadhesivematrices are formed such that the bioactive agent is released from thematrix upon contacting the matrix with a physiological medium. Thus, thebioadhesive formulations, according to some embodiments of the presentinvention, can be used for various bioadhesion applications, asdiscussed herein, while at the same time serving as a reservoir andvehicle for delivering a bioactive agent.

It is noted herein that while the incorporation of a bioactive agent inthe formulation may affect the characteristics of the formulation andthe characteristics of the resulting bioadhesive matrix, the bioadhesiveformulation and its corresponding matrix are designed to possess thedesired properties presented hereinabove while adding the capacity ofeluting bioactive agent(s) as discussed hereinbelow.

It is further noted herein that according to some embodiments of thepresent invention, the bioadhesive formulation and the correspondingmatrix containing no bioactive agent, is meant for use primarily for itsbioadhesive properties. In such embodiments, other that the amount andrate of releasing a bioactive agent, all other characteristics andtraits of an effective bioadhesive formulation/matrix described herein,as well as the optimal relative contents of the main constituents, applyfor a formulation not including a bioactive agent therein.

The term “incorporated”, as used in the context of a bioactive agent andthe bioadhesive formulation/matrix according to some embodiments of thepresent invention, is used synonymously with terms such as“sequestered”, “loaded”, “encapsulated”, “associated with”, “charged”and any inflection of these terms, all of which are used interchangeablyto describe the presence of the bioactive agent, as defined hereinbelow,within the formulation/matrix. A sequestered bioactive agent can eluteor be released from the matrix via, for example, diffusion, dissolution,elution, extraction, leaching, as a result of any or combination ofwetting, swelling, dissolution, chemical breakdown, degradation,biodegradation, enzymatic decomposition and other processes that affectthe matrix. A bioactive agent may also elute from the matrix without anysignificant change to the matrix′ structure, or with partial change.

As used herein, the phrase “bioactive agent” describes a molecule,compound, complex, adduct and/or composite that exerts one or morebiological and/or pharmaceutical activities. The bioactive agent canthus be used, for example, to relieve pain, prevent inflammation,prevent and/or reduce and/or eradicate an infection, promote woundhealing, promote tissue regeneration, effect tumor/metastasiseradication/suppression, effect local immune-system suppression, and/orto prevent, ameliorate or treat various medical conditions.

“Bioactive agents”, “pharmaceutically active agents”, “pharmaceuticallyactive materials”, “pharmaceuticals”, “therapeutic active agents”,“biologically active agents”, “therapeutic agents”, “medicine”,“medicament”, “drugs” and other related terms may be used hereininterchangeably, and all of which are meant to be encompassed by theterm “bioactive agent”.

The term “bioactive agent” in the context of the present invention alsoincludes diagnostic agents, including, for example, chromogenic,fluorescent, luminescent, phosphorescent agents used for marking,tracing, imaging and identifying various biological elements such assmall and macromolecules, cells, tissue and organs; as well asradioactive materials which can serve for both radiotherapy and tracing,for destroying harmful tissues such as tumors/metastases in the localarea, or to inhibit growth of healthy tissues, such as in current stentapplications; or as biomarkers for use in nuclear medicine andradio-imaging.

Bioactive agents useful in accordance with the present invention may beused singly or in combination, namely more than one type of bioactiveagents may be used together in one bioadhesive formulation, andtherefore be released simultaneously from the bioadhesive matrix.

In some embodiments, the concentration of a bioactive agent in theformulation ranges from 0.1 percents weight per volume to 10 percentsweight per volume of the total volume of said formulation, and even morein some embodiments. Higher and lower values of the content of thebioactive agent are also contemplated, depending on the nature of thebioactive agent used and the intended use of the bioadhesiveformulation/matrix.

When using the term “bioactive agent” in the context of releasing oreluting a bioactive agent, it is meant that the bioactive agent issubstantially active upon its release.

As discussed hereinbelow, the bioactive agent may have an influence onthe coupled gelatin-alginate matrix chemical and/or mechanicalproperties by virtue of its own reactivity with one or more of thematrix-forming components and/or the coupling agent, or by virtue of itschemical and/or physical properties per-se. It is therefore noted thatin general, the bioactive agent is selected suitable for beingincorporated into the bioadhesive formulation which affords the coupledgelatin gelatin-alginate bioadhesive matrix such that it can elute fromthe bioadhesive matrix in the intended effective amount and releaserate, while allowing the pre-curing bioadhesive formulation to exhibitdesired properties, as discussed herein, and while allowing theformulation to afford a bioadhesive matrix that exhibits the desiredproperties, as discussed herein.

As discussed and exemplified in the Examples section that follows, somebioactive agents may exhibit one or more functional groups which may besusceptible to the coupling processes taking place between the polymersand the coupling agent, and may therefore influence the characteristicsof the resulting matrix. For example, bioactive agents exhibiting acarboxylic group or a primary amine group may react with a couplingagent which is selected for its reactivity towards such functionalgroups. In such cases, in order to maintain desirable characteristics ofthe resulting matrix, some adjustments may be introduced to thebioadhesive formulation in terms of the type of ingredients and theirconcentrations.

A bioactive agent, according to some embodiments of the presentinvention, can be, for example, a macro-biomolecule or a small, organicmolecule.

According to some embodiments of the present invention, the bioactiveagent is a non-proteinous substance, namely a substance possessing nomore than four amino acid residues in its structure.

According to some embodiments of the present invention, the bioactiveagent is a non-carbohydrate substance, namely a substance possessing nomore than four sugar (aminoglycoside inclusive) moieties in itsstructure.

According to some embodiments of the present invention, the bioactiveagent is substantially devoid of one or more of the following functionalgroups: a carboxyl, a primary amine, a hydroxyl, a sulfhydroxyl and analdehyde.

The term “macro-biomolecules” as used herein, refers to a polymericbiochemical substance, or biopolymers, that occur naturally in livingorganisms. Amino acids and nucleic acids are some of the most importantbuilding blocks of polymeric macro-biomolecules, thereforemacro-biomolecules are typically comprised of one or more chains ofpolymerized amino acids, polymerized nucleic acids, polymerizedsaccharides, polymerized lipids and combinations thereof. Macromoleculesmay comprise a complex of several macromolecular subunits which may becovalently or non-covalently attached to one another. Hence, a ribosome,a cell organelle and even an intact virus can be regarded as amacro-biomolecule.

A macro-biomolecule, as used herein, has a molecular weight higher than1000 dalton (Da), and can be higher than 3000 Da, higher than 5000 Da,higher than 10 kDa and even higher than 50 KDa.

Representative examples of macro-biomolecules, which can be beneficiallyincorporated in the bioadhesive drug-eluting matrices described hereininclude, without limitation, peptides, polypeptides, proteins, enzymes,antibodies, oligonucleotides and labeled oligonucleotides, nucleic acidconstructs, DNA, RNA, antisense, polysaccharides, viruses and anycombination thereof, as well as cells, including intact cells or othersub-cellular components and cell fragments.

As used herein, the phrase “small organic molecule” or “small organiccompound” refers to small compounds which consist primarily of carbonand hydrogen, along with nitrogen, oxygen, phosphorus and sulfur andother elements at a lower rate of occurrence. In the context of thepresent invention, the term “small” with respect to a compound, agent ormolecule, refers to a molecular weight lower than about 1000 grams permole. Hence, a small organic molecule has a molecular weight lower than1000 Da, lower than 500 Da, lower than 300 Da, or lower than 100 Da.

Representative examples of small organic molecules, that can bebeneficially incorporated in the bioadhesive drug-eluting matricesdescribed herein include, without limitation, angiogenesis-promoters,cytokines, chemokines, chemo-attractants, chemo-repellants, drugs,agonists, amino acids, antagonists, anti histamines, antibiotics,antigens, antidepressants, anti-hypertensive agents, analgesic andanesthetic agents, anti-inflammatory agents, antioxidants,anti-proliferative agents, immunosuppressive agents, clotting factors,osseointegration agents, anti-viral agents, chemotherapeutic agents,co-factors, fatty acids, growth factors, haptens, hormones, inhibitors,ligands, saccharides, radioisotopes, radiopharmaceuticals, steroids,toxins, vitamins, minerals and any combination thereof.

Representative examples of bioactive agents suitable for use in thecontext of the present embodiments include, without limitation,analgesic, anesthetic agents, antibiotics, antitumor and chemotherapyagents, agonists and antagonists agents, amino acids,angiogenesis-promoters, anorexics, antiallergics, antiarthritics,antiasthmatic agents, antibodies, anticholinergics, anticonvulsants,antidepressants, antidiabetic agents, antidiarrheals, antifungals,antigens, antihistamines, antihypertensive agents, antiinflammatoryagents, antimigraine agents, antinauseants, antineoplastics,antioxidants, antiparkinsonism drugs, antiproliferative agents,antiprotozoans, antipruritics, antipsychotics, antipyretics, antisensesnucleic acid constructs, antispasmodics, antiviral agents, bile acids,calcium channel blockers, cardiovascular preparations, cells, centralnervous system stimulants, chemo-attractants, chemokines,chemo-repellants, chemotherapeutic agents, cholesterol, co-factors,contraceptives, cytokines, decongestants, diuretics, DNA, Drugs andtherapeutic agents, enzyme inhibitors, enzymes, fatty acids,glycolipids, growth factors, growth hormones, haemostatic andantihemorrhagic agents, haptens, hormone inhibitors, hormones,hypnotics, immunoactive agents, immunosuppressive agents, inhibitors andligands, labeled oligonucleotides, microbicides, muscle relaxants,nucleic acid constructs, oligonucleotides, parasympatholytics, peptides,peripheral and cerebral vasodilators, phospholipids, polysaccharides,proteins, psycho stimulants, radioisotopes, radiopharmaceuticals,receptor agonists, RNA, saccharides, saponins, sedatives, small organicmolecules, spermicides, steroids, sympathomimetics, toxins,tranquilizers, vaccines, vasodilating agents, viral components, viralvectors, viruses, vitamins, and any combination thereof.

The bioactive agent may be selected to achieve either a local or asystemic response. The bioactive agent may be any prophylactic agent ortherapeutic agent suitable for various topical, enteral and parenteraltypes of administration routes including, but not limited to sub- ortrans-cutaneous, intradermal transdermal, transmucosal, intramuscularadministration and mucosal administration.

One class of bioactive agents which can be encapsulated in thebioadhesive drug-eluting matrices, according to some embodiments of thepresent invention, is the class of analgesic agents that alleviate paine.g. NSAIDs, COX-2 inhibitors, opiates and morphinomimetics.

Another class of bioactive agents which can be incorporated in thebioadhesive drug-eluting matrices, according to some embodiments of thepresent invention, is the class of anesthetic agents. Another class ofbioactive agents which can be incorporated in the bioadhesivedrug-eluting matrices, according to some embodiments of the presentinvention, is the class of therapeutic agents that promote angiogenesis.Non-limiting examples include growth factors, cytokines, chemokines,steroids cell survival and proliferation agents.

Another class of bioactive agents which can be incorporated into thebioadhesive drug-eluting matrices, according to some embodiments of thepresent invention, especially in certain embodiments wherein tissueregeneration is desirable, and application involving implantable devicesand tissue healing, are cytokines, chemokines and related factors.

Non-limiting examples of immunosuppressive drugs or agents, commonlyreferred to herein as immunosuppressants, include glucocorticoids,cytostatics, antibodies, drugs acting on immunophilins and otherimmunosuppressants.

Non-limiting examples of haemostatic agents include kaolin, smectite andtranexamic acid.

It is noted herein that kaolin is an exemplary bioactive agent which hasa limited solubility in the bioadhesive formulation, and is thereforeadded in the form of a dry powder, and thus acts, at least to someextent, also as a filler in the bioadhesive formulation. This dualfunction, bioactive agent and filler, may characterize any additive orbioactive agent which are encompassed by embodiments of the presentinvention and are contemplated therewith.

Additional bioactive agents which can be beneficially incorporated inthe bioadhesive drug-eluting matrices, according to some embodiments ofthe present invention, include cytotoxic factors or cell cycleinhibitors and other agents useful for interfering with cellproliferation.

Additional bioactive agents which can be beneficially incorporated intothe bioadhesive drug-eluting matrices, according to some embodiments ofthe present invention, include genetic therapeutic agents and proteins,such as ribozymes, anti-sense polynucleotides and polynucleotides codingfor a specific product (including recombinant nucleic acids) such asgenomic DNA, cDNA, or RNA. The polynucleotide can be provided in “naked”form or in connection with vector systems that enhances uptake andexpression of polynucleotides. These can include DNA compacting agents(such as histones), non-infectious vectors (such as plasmids, lipids,liposomes, cationic polymers and cationic lipids) and viral vectors suchas viruses and virus-like particles (i.e., synthetic particles made toact like viruses). The vector may further have attached peptidetargeting sequences, anti-sense nucleic acids (DNA and RNA), and DNAchimeras which include gene sequences encoding for ferry proteins suchas membrane translocating sequences (“MTS”), tRNA or rRNA to replacedefective or deficient endogenous molecules and herpes simplex virus-1(“VP22”).

Additional bioactive agents which can be beneficially incorporated inthe bioadhesive drug-eluting matrices, according to some embodiments ofthe present invention, include gene delivery agents, which may be eitherendogenously or exogenously controlled.

Additional bioactive agents which can be beneficially incorporated intothe bioadhesive drug-eluting matrices, according to some embodiments ofthe present invention, include the family of bone morphogenic proteins(“BMP's”) as dimers, homodimers, heterodimers, or combinations thereof,alone or together with other molecules. Alternatively or, in addition,molecules capable of inducing an upstream or downstream effect of a BMPcan be provided. Such molecules include any of the “hedgehog” proteins,or the DNA's encoding them.

Additional bioactive agents which can be beneficially incorporated intothe bioadhesive drug-eluting matrices, according to some embodiments ofthe present invention, include chemotherapeutic agents.

Additional bioactive agents which can be beneficially incorporated intothe bioadhesive drug-eluting matrices, according to some embodiments ofthe present invention, include antibiotic agents.

Antiviral agents may include nucleoside phosphonates and othernucleoside analogs, AICAR (5-amino-4-imidazolecarboxamideribonucleotide) analogs, glycolytic pathway inhibitors, glycerides,anionic polymers, and the like.

Additional bioactive agents which can be beneficially incorporated intothe bioadhesive drug-eluting matrices, according to some embodiments ofthe present invention, include viral and non-viral vectors.

Additional bioactive agents which can be beneficially incorporated intothe bioadhesive drug-eluting matrices, according to some embodiments ofthe present invention, include steroidal anti-inflammatory drugs.

Additional bioactive agents which can be beneficially incorporated intothe bioadhesive drug-eluting matrices, according to some embodiments ofthe present invention, include anti-oxidants.

Additional bioactive agents which can be beneficially incorporated intothe bioadhesive drug-eluting matrices, according to some embodiments ofthe present invention, include vitamins.

Additional bioactive agents which can be beneficially incorporated intothe bioadhesive drug-eluting matrices, according to some embodiments ofthe present invention, include hormones.

Additional bioactive agents which can be beneficially incorporated intothe bioadhesive drug-eluting matrices, according to some embodiments ofthe present invention, include cells of human origin (autologous orallogeneic), including stem cells, or from an animal source(xenogeneic), which can be genetically engineered if desired to deliverproteins of interest.

Drug Release Profile:

The rate of release of the bioactive agent, or the drug release profilefrom the matrix, serving as its reservoir, can be controlled by variousfactors, such as the relative concentrations of the constituents in thebioadhesive formulation described herein.

A typical drug delivery mechanism, relevant in the context of thepresent embodiments, consists of a reservoir containing a predeterminedand exhaustible amount of the drug, and an interface between the drug'sreservoir and the physiological environment. Typically, the drug releasecommences at the initial time point when the reservoir is exposed to thephysiological environment, and follows typical diffusion-controlledkinetics with additional influences effected by water uptake (swelling),disintegration and biodegradation of the matrix and the likes.

It is noted that contrary to synthetic polymers that are used in theformulation that affords other drug-eluting matrices, such aspoly-lactic acid (PLA) and poly-glycolic acid (PGA), thegelatin-alginate coupled matrix is not water degradable, neither thenatural polymers, nor the coupling product amide bond. In a live subjectbody this matrix will pass degradation through enzymatic activity, whichwould further affect the drug release rate.

A “drug release profile” is a general expression which describes thetemporal concentration of a drug (a bioactive agent) as measured in aparticular bodily site of interest as a function of time, while theslope of a concentration versus time represents the rate of release atany given time point. A drug release profile may be sectioned into ratedependent periods whereby the rate is rising or declining linearly orexponentially, or staying substantially constant. Some of the typicallysought rates include the burst release rate and the sustained releaserate.

The phase “burst release”, as used herein, is consistent with a rapidrelease of a drug into the bodily site of interest, and is typicallyassociated with an exponential increase of the drug's concentration,growing from zero to a high level at a relatively short time. Typically,the burst release section of the drug release profile ends briefly andthen gradually changes to a plateau, or a sustained release section inthe release profile.

The phrase “sustained release”, as used herein, refers to the section ofthe drug release profile which comes after the burst release part, andis typically characterized by constant rate and relative long duration.

The main differences between the burst and the sustain parts of arelease profile are therefore the rate (slope characteristics) andduration, being exponential and short for the burst release, and linearand long for the sustained release; and both play a significant role indrug administration regimes. In most cases, the presence of both a burstrelease section and a sustained release section is unavoidable and stemsfrom chemical and thermodynamic properties of the drug deliverymechanism.

In the context of embodiments of the present invention, the phrase “highburst release” is an attribute of a drug-eluting bioadhesive matrix, asdescribed herein, which refers to the amount of drug that is beingreleased from the matrix during the initial stage of exposure of thematrix to the environment of its action (e.g., physiologicalenvironment), wherein the amount is in excess of 20% of the total amountcontained in the matrix and the initial stage is regarded as the firstsix hours from exposure.

In some embodiments of the present invention, “high burst release”describes an attribute of a drug-eluting bioadhesive matrix, asdescribed herein, in which 30%, 40%, 50%, 60% and even higherpercentages of the bioactive agent (drug) are released during the first6 hours of exposing the matrix to a physiological medium. Any valuebetween 20% and 100% of the bioactive agent (drug) are contemplated.

Accordingly, the phrase “low burst release” refers to drug-elutingbioadhesive matrices wherein less than 20% of the contained drug isreleased within the first six hours of exposure.

In some embodiments of the present invention, “low burst release”describes an attribute of a drug-eluting bioadhesive matrix, asdescribed herein, in which 15%, 10%, 5% and even lower percentages ofthe bioactive agent (drug) are released during the first 6 hours ofexposing the matrix to a physiological medium. Any value between 20% and1% of the bioactive agent (drug) are contemplated.

In general, and according to some embodiments of the present invention,at least 20 percents of the bioactive agent are released to thesurrounding physiological medium within 6 hours of contacting theformulation/matrix with physiological medium.

According to some other embodiments of the present invention, no morethan 20 percents of the bioactive agent are released to the surroundingphysiological medium within 6 hours of contacting the formulation/matrixwith physiological medium.

Preparation of the Bioadhesive Formulation and Matrix Formation:

The bioadhesive formulations presented herein, either containing or notcontaining a bioactive agent, are prepared by mixing all the ingredientstogether into a single concoction, at least in the sense of theformulation which is capable of curing.

The single concoction can be formed ex vivo, in vitro or in situ, namelythe formulation can be in the form of two or more sub-formulations keptseparately, or as a set of dry powders and a pre-measured amount ofsolvent (water) kept separately, as discussed hereinbelow, which arecombined to form the single concoction by one of the following manners.

In vitro means that the formulation as a single concoction is formed bymixing (e.g., in a vial) all the components of the formulation, as theseare defined, described and exemplified herein, prior to applying theformulation onto the object(s) to be bonded.

In situ means that the formulation as a single concoction is formed byapplying one sub-formulation on one object, and another sub-formulationon another object, and adjoining the objects together to form the singleconcoction at the site of adhesion; or by applying one sub-formulationon an object and thereafter applying another sub-formulation on the sameobject.

When applied to animated objects, in vitro corresponds to ex vivo, andin situ corresponds to in vivo.

In any eventuality, the formulation is kept under conditions where it issubstantially unable to cure.

According to an aspect of some embodiments of the resent invention,there is provided a method of forming a bioadhesive matrix, which iseffected by curing the bioadhesive formulation as described herein.

As used herein, the term “curing” includes an active procedure such assubjecting the formulation to certain conditions (e.g., heating and/ormixing, shear forces, etc.), as well as a passive procedure, whichinvolved allowing the curing time to elapse.

In some embodiments, the method further comprises, prior to the curing,mixing the components of the formulation, namely, mixing theherein-described sub-formulations or mixing the herein-describedpowder(s) with the appropriate solvent(s).

As discussed hereinabove, the mixing can be effected ex vivo, in vivo,in vitro or in situ.

Use of the Bioadhesive Formulations and Matrices:

As discussed herein, the bioadhesive formulations presented herein,either containing or not containing a bioactive agent, are used to forma bioadhesive matrix, being either a drug-eluting or non-drug-elutingmatrix, which is useful in many medical and paramedical applications. Asdiscussed above, the bioadhesive matrices, according to some embodimentsof the present invention, are useful, for example, in replacing orreinforcing surgical sutures and staples.

Hence, according to some embodiments, the formulations presented herein,either incorporating a bioactive agent or not, are identified for use inbonding objects to one another, wherein at least one of these objects isa biological object, as these terms are defined and discussed herein.

In general, the bioadhesive formulation presented herein can be used inthe manufacturing of a product intended for bonding objects, at leastone of which is a biological object.

Thus, the phrase “biological object”, as used herein, refers to anyviable/live part of an animal or plant, including a single live animalspecimen. A live or viable biological object or tissue is defined as anymajor or minor part of a plant or animal that is still viable or aliveand substantially kept in a physiological environment in order to stayviable or alive. Non-limiting examples of biological objects include anyplant or animal, viable tissue samples, skin tissue, bone tissue,connective tissue, muscle tissue, nervous tissue and epithelial tissue.Also encompassed are edges of incisions made in an organ, such as skin,muscle, internal organ in any bodily site of an organism.

Inanimate objects are objects which cannot be revived, grafted,proliferate or otherwise show any signs of life as defined medically,and include objects of synthetic and/or biological origins. Theseinclude, for example, patches, bone-replacement parts, pace makers,ports and vents and any other medical device that required affixing andimmobilization to a viable biological object as defined herein.

According to some embodiments of the present invention, inanimatebiological objects can be made partially or entirely from animal orplant materials and products, or partially or entirely from syntheticsubstances. While the bioadhesive formulation is designed for use in oron viable biological objects, it is noted herein that is can be usedeffectively to bond biologic or synthetic inanimate objects like anyadhesion agent or glue.

It is noted herein that the term “object” is meant to encompass one ormore parts or portions of the same object, thus closing an incision bybonding the two sides of the incision in a tissue or an organ, by usingthe herein-described formulation, can be regarded as either bonding oneobject (the tissue or organ) or two objects (the two sides of theincision).

According to some embodiments, the drug-eluting matrix, resulting from abioadhesive formulation which incorporates a bioactive agent, is usedsolely for its drug-eluting and drug-delivery faculties regardless ofits bioadhesive faculty. Such a matrix can serve, for example, as a drugdepot, and can be adhered to an organ or tissue where the release of thedrug is beneficial (without bonding thereto another object).

Methods of Forming Bioadhesive Matrices and Bonding Objects:

According to an aspect of embodiments of the present invention, there isprovided a method of forming the herein-described bioadhesive matrixusing the herein-described bioadhesive formulation, the method iseffected by curing the formulation.

According to some embodiments of the present invention, the bioadhesivematrix, being formed by curing a bioadhesive formulation, containing abioactive agent or not, is formed on an object and/or between two ormore objects, wherein at least one of the objects is a biological objectas described herein.

The formation of the herein-described bioadhesive matrix on an object iseffected by applying the herein-described bioadhesive formulation on theobject and curing the formulation. The formation of the matrix betweentwo or more objects is effected by applying the formulation on one ormore of the objects, for example, on the parts of their surface that isintended for bonding, and thereafter adjoining the objects. By adjoiningthe objects it is meant that at least parts of the surface of eachobject are put in contact.

In some embodiments of the invention, adjoining the object is furtheraccompanied by using force and/or other means of fastening the objectsand keep the objects adjoined (prevent the bonded surfaces fromseparating) one to the other at least until the formulation cures tosuch extent that the object are bonded and can be let go.

Accordingly, there is provided a method of bonding at least two objectsto one another, which is effected by:

applying the bioadhesive formulation presented herein onto at least oneof the objects; and

adjoining the objects (so as to form a contact therebetween), optionallywhile fastening the object to one another.

It is noted that the action of adjoining is effected so as to formcontact between the object, while fastening is used in order to allowthe formulation to cure and form the matrix between the objects, therebybonding the objects to one-another.

According to some embodiments of the present invention, the bioadhesiveformulations and matrices presented herein can be used to hold bodytissues together after an injury or surgery, as well as in many surgicalprocedures including, but not limited to incision closure, cornealperforations, episiotomy, caesarian cases, cleft lip, skin and bonegrafting, tendon repair, hernia, thyroid surgery, periodontal surgery,gingivectomy, dental implants, oral ulcerations, gastric varices woundsof internal organs such as liver and pancreas, attachment andimmobilization of external and internal medical devices and more.

Means for Storing, Preparing and Applying Bioadhesive Formulations:

As presented herein, the bioadhesive formulation according to someembodiments of the present invention consists of two or more majorpolymeric components and a coupling agent that forms crosslinking bondsbetween the major polymeric components. Since the coupling/crosslinkingreaction occurs upon contacting the polymeric components with thecoupling agents, these two groups of components, namely the polymers andthe coupling agents, should be kept separated until there is a need toapply the formulation. It is therefore noted that the formulation can bekept in any number of separated parts, at least until it is being mixedinto a single concoction comprising all ingredients prior to itsapplication and use.

Hence, according to some embodiments of the present invention, thepre-curing bioadhesive formulation presented herein is formed bycontacting a sub-formulation A that contains primarily gelatin, alginateand an optional bioactive agent, with a sub-formulation B containsprimarily one or more coupling agents.

Alternatively, the pre-curing bioadhesive formulation can be keptindefinitely as dry mixture of powders of pre-measured amounts of eachof its constituents, including the polymers, the coupling agents,various additives and the bioactive agents, permitting each can bereconstituted by dissolution in water. Alternatively only some of theconstituents are kept as dry mixture of powers while other constituentsare kept as separate solutions or powders. It is noted that any form oflong-term storage of the bioadhesive formulation is contemplated as longas the coupling/crosslinking reaction is prevented from commencinguncontrollably.

Alternatively, according to some embodiments of the present invention,sub-formulation A and sub-formulation B are each kept in sealedcompartments, each compartment may serve as a storage vessel, or as areservoir of an integrated applicator (e.g., configured for applyingpastes and thick liquids) adapted for applying the bioadhesiveformulation presented herein.

Hence, according to another aspect of some embodiments of the presentinvention, there is provided a kit for storing, preparing and/orapplying the pre-curing bioadhesive formulation presented herein, whichincludes at least two compartments, such as a first compartment and asecond compartment, wherein the first compartment containssub-formulation A, as presented hereinabove, and the second compartmentcontains sub-formulation B. As long as these two compartments are keptsealed and under acceptable storage conditions, the bioadhesiveformulation will not cure or disintegrate.

In some embodiments, the kit includes at least two compartments, eachcontaining the constituents corresponding to the particularsub-formulation, which have been pre-dissolved in a solvent to aspecific concentration such that mixing the two sub-formulations resultsin a bioadhesive formulation as described herein.

Alternatively, the kit includes one or more compartments, eachcontaining a pre-measured amount of a dry powder of one or moreconstituent of the bioadhesive formulation, and a separate compartmentcontaining a pre-measured amount of the solvent, such that mixing thepowder(s) and the solvent results in a bioadhesive formulation asdescribed herein.

The kit may further include mixing and stirring tools, bowls,applicators, freshness indicators, tamper-proof measures and printedmatter for instructions for the user.

The kit may include a device, an applicator or a dispenser for expellingmeasured amounts of each sub-formulation controllably and optionallysynchronously, each of which is dispensed from the individualcompartment serving as a cartridge of the individual sub-formulation.

Hence, according to another aspect of some embodiments of the presentinvention, there is provided an integrated dual chamber dispenser foruse in applying the bioadhesive formulation presented herein, whichincludes a dual barrel cartridge assembly with a joint delivery portwith a mount therein for coupling with a mixing tube. Integratedapplicators suitable for applying the bioadhesive formulation presentedherein, may follow the design of any applicator for two-part chemistryadhesives which require efficient dispensing under controlled and safeconditions of two sub-formulations from separate compartments.

Exemplary two-part chemistry adhesive applicators, which can be used tomix, dispense and apply the bioadhesive formulations presented hereinare disclosed, for example, in U.S. Patent Application Publication Nos.2007/0289996 and 2011/0248045, and in U.S. Pat. Nos. 4,979,942,5,082,147, 6,732,887, 7,530,808, 7,635,343, 7,699,803, 8,074,843, all ofwhich are incorporated herein by reference as if full set forth herein.

As used herein the term “about” refers to ±10%.

The terms “comprises”, “comprising”, “includes”, “including”, “having”and their conjugates mean “including but not limited to”.

The term “consisting of” means “including and limited to”.

The term “consisting essentially of” means that the formulation,composition, method, matrix or structure may include additionalingredients, steps and/or parts, but only if the additional ingredients,steps and/or parts do not materially alter the basic and novelcharacteristics of the claimed formulation, composition, method, matrixor structure.

As used herein, the singular form “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention maybe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicate number and asecond indicate number and “ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and aremeant to include the first and second indicated numbers and all thefractional and integral numerals therebetween.

As used herein the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Various embodiments and aspects of the present invention as delineatedhereinabove and as claimed in the claims section below find experimentalsupport in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions illustrate some embodiments of the invention in a nonlimiting fashion.

Example 1 Materials and Methods

Materials:

An exemplary basic bioadhesive matrix, according to some embodiments ofthe present invention, was prepared from an exemplary bioadhesiveformulation containing the following ingredients:

Gelatin—

In the example presented herein gelatin type A from porcine skin (90-100bloom) was purchased from Sigma-Aldrich. For the adhesive mechanismstudies, three types of gelatin “type A” from porcine skin withdifferent Bloom numbers (90-100, 175 and 300) were used.

Alginate—

In the example presented herein alginic acid sodium salt (viscosityabout 250 cps, 2% (25° C.) was purchased from Sigma-Aldrich. For theadhesive mechanism studies, alginic acid sodium salts with low (LV) andhigh viscosity (HV) of 100-300 cP (0.1-0.3 Pa-sec) and more than 2,000cP (2 Pa-sec), 2% (25° C.) respectively were used.

EDC—

N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride waspurchased from Sigma-Aldrich.

Bupivacaine hydrochloride was purchased from Sigma-Aldrich. It is alocal anesthetic used for peripheral nerve block, infiltration, andsympathetic, caudal or epidural block.

Ibuprofen sodium salt was purchased from Sigma-Aldrich. It—is a commonlyused non-steroidal anti-inflammatory drug used in the treatment of pain,fever, dysmenorrhea, osteoarthritis, rheumatoid arthritis, and otherrheumatic and nonrheumatic inflammatory disorders, and vascularheadaches.

Synthetic hydroxylapatite (also called hydroxyapatite, HA), in the formof a powder,) and beta-tricalcium phosphate (β-TCP) in the form ofsintered powder, were purchased from Sigma-Aldrich, Rehovot, Israel.

Bioadhesive Preparation:

Drug-eluting bioadhesive preparation is generally based on dissolvingvarious amounts of gelatin, alginate and a bioactive agent as powders orstock solutions in distilled water, under heating condition of up to 60°C. on a hot plate. Due to the high viscosity of the final solution,stirring is effected by an agitator such as Fisher's Vortex Genuine 2™stirring device.

Briefly, a pre-measured amount of a bioactive agent is dissolved indistilled water. Thereafter a pre-measured amount of alginate is addedinto the clear and transparent homogenous solution of the bioactiveagent. Once a bright yellow homogenous viscous solution is afforded, apre-measured amount of gelatin i added thereto to afford agelatin-alginate-active agent mixture, also referred to herein assub-formulation A. Various pre-measured amounts of the coupling agentEDC are dissolved in a separate distilled water tube to affordsub-formulation B, which is added to sub-formulation A just prior to thetime of bioadhesive use.

Pristine bioadhesive matrices not sequestering any bioactive agent areprepared from a bioadhesive formulation without a bioactive agent,similarly to the procedure above with the exception that alginate isfirst dissolved in pure distilled water.

Viscosity Measurements:

Since the initial viscosity of the bioadhesive formulation is affectedmainly by the viscosity of the gelatin-alginate solution, viscositymeasurements were conducted prior to the addition of EDC. The viscositymeasurements were performed using a controlled stress rheometer (modelAR2000, TA Instruments Ltd.), fitted with a cone-and-plate geometry (4°cone angle, 40 mm diameter, 400 μm gap), at a constant temperature of37° C. and a constant shear rate of 10 Hz in order to investigate therelations between the adhesive's initial viscosity and its bondingstrength.

Soft Tissue Bonding Strength Measurements:

In vitro soft tissue bonding strength measurements of variousbioadhesive matrices, according to some embodiments of the presentinvention, were performed in order to examine the effect of thebioactive agent's type and concentration in the bioadhesive formulationon the bonding strength, namely, the ability to bind two soft tissuesamples together.

Porcine skin (purchased from Kibbutz Lahav, Israel) was used assoft-tissue model for the bonding test. The porcine skin was cut into 2cm×2 cm square-shape pieces, using a scalpel, and the pieces were keptin −20° C. until use. At time of use, the porcine skin pieces werethawed in air and their epidermis side was attached firmly withsuper-glue to 1 cm height brass testing holders with a matching surfacearea. The testing holders were connected to brass grip tabs of 2 cm×0.4cm×5 cm.

0.1 ml of sub-formulation A (the gelatin-alginate mixture) was spreaduniformly and equally on the dermis side of two porcine skin pieceshaving their epidermis side attached to the testing holders, aspresented hereinabove. Thereafter, 0.04 ml of sub-formulation B (EDCsolution) was mixed with sub-formulation A on one of the skin pieces toafford a pre-curing bioadhesive formulation, which turns into abioadhesive matrix once cured. Subsequently, the holder attached to theother piece was placed above the first holder, in a way that a firmcontact was established between the dermis sides of the both skinpieces. The two holders were then pressed together in a constant forceof 1.25 N and were placed in a static incubator (Heraeus, B12) at 37° C.and 100% humidity environment for 30 minutes. Five identical coupledskin samples were prepared using each tested bioadhesive formulation.

After 30 minutes of incubation, the bonding strength of the bioadhesivematrix was measured in tension at room temperature using a 5500 InstronUniversal Testing Machine (Instron engineering Corp.) and a 10 N loadcell. The two holder adjoined by the coupled skin pieces were strainedunder constant velocity of 2 mm/min until separation was achieved andthe result was recorded in kilo-pascal (KPa). The mechanical testingprocedure was inspired by standard test method ASTM F-2258-03. Theboning strength was defined as the maximum strength in the stress-straincurve, measured by the Instron Merlin software.

Drug-Eluting Studies:

In vitro release studies were preformed in order to examine the effectof the bioadhesive components on the release kinetics of two types ofbioactive agents in the form of the anesthetic drugs bupivacaine andibuprofen. Five repetitions were carried out for each formulation.

0.1 ml of the tested sub-formulation A (gelatin-alginate mixture) wasinjected, using a 1 ml syringe without needle, into 6 mm×6 mm×3 mmrectangular silicone molds. Immediately thereafter, 0.04 ml ofsub-formulation B (EDC aqueous solution) was mixed with sub-formulationA to form a pre-curing bioadhesive formulation (140 μl) in the mold. Thewet bioadhesive matrix samples were pressed out of their silicone moldsa few minutes later, after the bioadhesive formulation was fully cured,and air-dried in a chemical hood.

The air-dried bioadhesive matrix's cuboids were put in plastic testtubes, and 1 ml of sterile PBS buffer at pH 7 was add to each test tubeand used as a release medium. Sodium azide (0.02% w/v) was added to themedium in order to prevent microbial contamination. After adding themedium, the test tubes (with the bioadhesive samples inside) were sealswith plastic caps and were placed in a static incubator (Heraeus, B12)at 37° C. for 14 days.

At specific time points during the experiment, namely 6 hours, 1, 2, 3,7 and 14 days, the entire medium was removed from the test tubes andreplaced with an equal volume of fresh PBS buffer solution and 0.02% w/vsodium azide. The removed medium was transferred into glass HPLC vialsand kept at −20° C. until analyzed. For the determination of the drugrelease kinetics, the concentrations of the drugs in the removed mediumat each time point were evaluated using an HPLC system.

The HPLC analyses of the samples containing bupivacaine were performedusing a Jasco HPLC system equipped with a UV 2075 plus detector that wasset on 210 nm, and a reverse phase column (ACE 5 C18, inner diameterd=4.6 mm, length=250 mm), kept at 40° C. The mobile phase consisted of amixture of PBS (pH 3.3) and acetonitrile mixture (72:28%, v/v) at a flowrate of 1.5 ml/min with a quaternary gradient pump (PU 2089 plus)without gradient. 1-100 μl samples were injected with an auto sampler(AS 2057 Plus). The area of each eluted peak was integrated usingEZstart software version 3.1.7, using a calibration curve.

The HPLC analyses of the samples containing ibuprofen were performedusing a Jasco HPLC system equipped with a UV 2075 plus detector that wasset on 220 nm, and a reverse phase column (ACE 5 C18, inner diameterd=4.6 mm, length=250 mm), kept at 40° C. The mobile phase consisted of amixture of PBS (pH 3.3) and acetonitrile mixture (40:60%, v/v) at a flowrate of 2 ml/min with a quaternary gradient pump (PU 2089 plus) withoutgradient. 1-100 μl samples were injected with an auto sampler (AS 2057Plus). The area of each eluted peak was integrated using EZstartsoftware version 3.1.7, using a calibration curve.

14 days after the in vitro drug elution process has started, thebioadhesive samples were immersed in “trypsin A” solution at 37° C. for4 hours, in order to dissolve the samples and to extract the drug thatwas not released during the first 14 days. The dissolved solutions werefiltered using a disposable filer unit (Whatman, 0.2 μm), injected intothe HPLC system and analyzed according to the protocols mentionedhereinabove in order to determine the amount of the drug remainders.

Water Uptake:

Two hours after mixing the pre-curing bioadhesive formulation containingprimarily gelatin and alginate with EDC, the cured bioadhesive matrixwas weighed and a dry weight W_(dry) was recorded. Thereafter fivemilliliters of PBS buffer solution (pH 7) was added thereto. The platewith the bioadhesive matrix sample was inserted to an incubator(Heraeus, B12) at 37° C. and 100% humidity for 0.5, 1, 2, 5, 24 and 48hours.

When the allotted time expired the PBS solution was removed from theplate and the swollen bioadhesive matrix sample was weighed and the wetweight W_(wet) was recorded. The water uptake (swelling) ratio wascalculated according to the formula (W_(wet)−W_(dry))×100/W_(dry).

The drug release rate can be affected by the swelling of a drug-loadedhydrogels. Air-dried drug free bioadhesive cuboids were placed inplastic test tubes and were soaked in 1 ml drug release medium (PBS pH 7and 0.02% sodium azide) and placed in a static incubator (Heraeus, B12)at 37° C. for 6 hours. Weight values of the samples were taken beforesoaking (W_(dry1)), after 6 hours of soaking (W_(wet)) and afterair-drying the soaked samples (W_(dry2)). The water uptake wascalculated as (W_(wet)−W_(dry2))×100/W_(dry1).

Cytotoxicity Test:

Fibroblast cells (14^(th) passage) were thawed and cultured in 75 mm²flasks with culture medium at 37° C., humidified atmosphere and 5% CO₂.The cells were cultured with modified Eagle's medium supplemented with10% fetal bovine serum, 1% L-glutamine and 0.1%penicillin-streptomycin-nystatin. At confluence of 70% (at passages16-19) the cells were separated using incubation for 3 minutes with 1 mltrypsin A, the free cells were added to the culture medium and seeded ina 6 well plate.

When confluence of 70% was reached the medium was replaced by 2 ml ofmedium containing 10% v/v Alamar blue for 4 hours of incubation.Thereafter, two 100 μL samples were taken from each well and inserted toa 96 well plate. The plate was inserted for analysis at aspectrophotometer (Spectra max 340 PC384, Molecular Devices) at 570 and600 nm.

Two experiments were carried out, at first each component of thebioadhesive were inserted separately to a test tube containing 4 mlmedium and incubated for 24 hours and then that medium was added to thecells instead of the medium containing Alamar Blue. In the secondexperiment, the medium with Alamar Blue was replaced with 4 ml of freshmedium and 0.1 ml of bioadhesive was added to the medium and incubatedfor 24 hours followed by another reading using Alamar Blue.

The reduction of Alamar Blue is an indication of changes in cellproliferation; hence the results were compared to the initial readingwithout the tissue adhesive. The reduction of Alamar Blue's absorptionwas calculated using the manufacturer's protocol, namely Alamar Bluereduction=(ε₆₀₀A^(t)570−ε₅₇₀A^(t) ₆₀₀)/(ε₅₇₀A^(c) ₆₀₀−ε₆₀₀A^(c) ₅₇₀),wherein ε₅₇₀ and ε₆₀₀ are the molar extinction coefficients of oxidizedAlamar Blue at 570 nm and 600 nm respectively, A^(t) and A_(c) are theabsorbance of test and control respectively at 570 nm and 600 nmrespectively, whereas the control is taken as the medium with Alamarblue but without any cells.

Microstructure Characterization:

The microstructure of drug-loaded bioadhesive matrices, according tosome embodiments of the present invention, was investigated in order tocharacterize the dispersion of different drugs in the bioadhesivematrix, and to examine the effect of the microstructure on the drugrelease profiles and bonding strength. For this purpose, air-drieddrug-loaded bioadhesive cuboids of about 24×24×3 mm, prepared from 420μl of a bioadhesive formulation according to embodiments of the presentinvention, were freeze fractured and their cross section was observedusing an environmental scanning electron microscope (Quanta 200 FEGESEM) in a high vacuum mode, with an accelerating voltage of 10 kV. Themean diameter of the drug crystals and aggregates was analyzed using theSigma Scan Pro software.

Hard Tissue Bonding Strength Measurements:

Cortical portions of bovine femurs (purchased at a local abattoir) wereused as a hard tissue model in order to evaluate the effect of theaddition of fillers to the bioadhesive formulation on the bondingstrength of the resulting bioadhesive matrix.

Femur bone samples were sawed into 2×2×0.2 cm cuboid specimens using a“FMB-minor” portable band saw. The specimens were attached firmly tometal testing holders with a matching surface area.

The bonding strength measuring system was based on the system used forsoft tissue adhesion as presented herein, and had similar dimensions.

140 μl of the bioadhesive formulation containing various concentrationsof the tested fillers, were spread uniformly on the exposed side of twofemur specimens. The specimens were then immediately attached to eachother by applying a load of 7.5 N and placed in a 37° C. and 100%humidity environment. After 30 minutes, the bonding strength wasmeasured in tension mode at room temperature using a 5500 Instronuniversal testing machine (Instron Engineering Corp.) and a 2 kN loadcell.

The two parts of the femur joint were strained at a constant velocity of2 mm per minute until separation was achieved.

The mechanical testing procedure was inspired by the standard testmethod ASTM F-2258-03. The bonding strength was defined as the maximumstrength in the stress-strain curve, measured by the Instron Merlinsoftware.

Example 2 Background Art

Formulations based on the teaching of Sung et al. [J. Biomed. MaterialsRes., 46(4), p. 520-530, 1999], were prepared for comparison with thebioadhesive formulations and matrices according to some embodiments ofthe present invention.

Briefly, stock solution were prepared by pre-weighed amount of alginatewas dissolved in distilled water at 50° C. and once the solution clearedpre-weighed amount of gelatin was added thereto and stirred until thesolution became clear. The resulting mixture contained 600 mg/ml gelatinand 30 mg/ml alginate. The coupling agent EDC was dissolved in distilledwater to afford a 10 mg/ml solution and a 20 mg/ml solution.

Testing of the resulting formulation was performed as follows. Shortlyprior to use, 0.1 ml gelatin-alginate stock solution was mixed with0.035 ml EDC stock solution to afford a final concentration of 445 mg/mlgelatin, 22 mg/ml alginate and 2.6 mg/ml EDC. This mixture was preparedon a piece of pig skin and thereafter a second piece of skin was laidover the formulation and the coupled skin pieces were put under pressureof 0.129 kg for 30 minutes in a moist incubator (100% humidity, 37° C.).The coupled skin pieces were subjected to mechanical testing protocol aspresented hereinabove, and exhibited a result of 4436±1361 Pa. It isnoted that the resulting formulation was difficult to apply on the skinpiece due to too rapid gelation at room temperature.

In another attempt to reproduce the formulation reported by Sung et al.a formulation having a final concentration of 600 mg/ml gelatin, 30mg/ml alginate and 20 mg/ml EDC, prepared from a stock solution of 840ml/ml gelatin and 42 mg/ml alginate in water. This formulation wasimpossible to apply regardless of the coupling agent concentration.

Example 3 Bonding Strength

Several series of measurements were performed in order to elucidate theeffect of each component of the bioadhesive formulation on the resultingbioadhesive matrix′ bonding strength, expressed in kilo-pascal (KPa).The effects of the gelatin, alginate and coupling agent EDCconcentrations are presented in Table 1, FIG. 1 and FIG. 2.

Table 1 presents the results of the bonding strength tests as conductedon four series of samples divided by varying parameter, wherein alginatevaries in Series 1, 2 and 3 while gelatin varies across Series 1-3, andEDC varies in Series 4 while gelatin and alginate are constant.

TABLE 1 Gelatin Alginate EDC Bonding Formu- concentration concentrationconcentration strength lation (mg/ml) (mg/ml) (mg/ml) (KPa) Series 1 10020 4 2.29 ± 0.29 100 40 4 2.22 ± 0.38 100 60 4 2.57 ± 0.34 Series 2 15020 4 3.17 ± 0.48 150 40 4 4.12 ± 0.55 150 60 4 2.58 ± 0.3  Series 3 20020 4 4.99 ± 0.43 200 40 4  5.5 ± 0.29 200 60 4  3.2 ± 0.36 Series 4 20040 0 2.79 ± 0.44 200 40 4 2.81 ± 0.45 200 40 10 5.02 ± 0.57 200 40 155.52 ± 0.91 200 40 20 9.84 ± 0.85

As can be seen in Table 1, FIG. 1 and FIG. 2, the bonding strength isincreased with the increase in gelatin concentration, and the highestbonding strength was observed for alginate concentration is about 40mg/ml. An increase in the EDC concentration resulted in an increase inthe bonding strength.

A bioadhesive formulation, comprising gelatin at a concentration of 200mg/ml, alginate at a concentration of 40 mg/ml and EDC at aconcentration of 20 mg/ml, was selected to represent an exemplarybioadhesive formulation for the studies presented hereinbelow. Thisbioadhesive formulation resulted in a bioadhesive matrix exhibitingbonding strength of approximately 10 KPa, which is four-times strongerthan that of Evicel™ (a fibrin sealant produced by Johnson and Johnsonwith a bonding strength of approximately 2.5 KPa as measured using thesystem and protocol presented hereinabove).

The effect of time on the bonding strength of the selected bioadhesiveformulation is presented in FIG. 3.

As can be seen in FIG. 3, the bonding strength only slightly changesduring the first 5 hours, but after 10 hours there is a significantincrease in the bonding strength and it reaches approximately 24 KPa.The later may explained by some dehydration of the skin. The resultsshow that this exemplary bioadhesive matrix maintains sufficientstrength for at least 10 hours.

Two hours after mixing the components of the bioadhesive formulation, asample of the resulting bioadhesive matrix was immersed in an aqueousmedium for 24 hours so as to evaluate its water uptake kinetics to givesan indication for swelling and the results are presented in FIG. 4.

As can be seen in FIG. 4, the water uptake increases with immersion timelinearly, while it is relatively low during the first 5 hours. Thisresult is desirable for tissue bioadhesive applications.

Example 4 Elution of Bioactives

Analyzing the influence of the components of the bioadhesiveformulations, according to some embodiments of the present invention, onvarious drug release profiles from the corresponding bioadhesive matrix,allows not only designing a controlled release product that fits thespecific therapeutic demands of the bioadhesive, but also providesinsights on the principles of drug release kinetics from a bioadhesivematrix.

Effect on Bonding Strength:

Two types of drug-eluting samples of bioadhesive matrices were examined:bupivacaine-loaded bioadhesive matrices and ibuprofen-loaded bioadhesivematrices.

A formulation containing gelatin (200 mg/ml), alginate (40 mg/ml) andEDC (20 mg/ml) was chosen an exemplary bioadhesive formulation. Thebonding strength in tension was measured for matrices containing threedifferent drug concentrations (loads), 1% w/v, 2% w/v and 3%, and theresults are presented in FIG. 5 and FIG. 6, and in Table 2 hereinbelow.

TABLE 2 Gelatin Alginate EDC Drug Bonding content content contentcontent strength [mg/ml] [mg/ml] [mg/ml] [% w/v] [kPa] Drug-free 200 4020 0 9.84 ± 1.91 bioadhesive Bupivacaine 200 40 20 1 10.10 ± 1.73 loaded 2 13.71 ± 2.21  bioadhesives 3 15.01 ± 1.92  Ibuprofen 200 40 201 6.10 ± 1.12 loaded 2 4.37 ± 1.13 bioadhesives 3 4.47 ± 0.78

As can be seen in Table 2, FIG. 5 and FIG. 6, bioadhesive matricesincorporating bupivacaine and ibuprofen showed an opposite effect ontheir bonding strengths. While bupivacaine has a positive effect ofincreasing the bonding strength of the bioadhesive, ibuprofen decreasedthe bonding strength.

Drug Release Profile:

The effects of the components of the bioadhesive formulations and drugcontent on the drug release profiles were examined in two types ofdrug-eluting bioadhesive matrices as presented hereinabove.

A reference bioadhesive formulation of 200 mg/ml gelatin, 40 mg/mlalginate and 20 mg/ml EDC containing 3% w/v bioactive agent, bupivacaineor ibuprofen, was chosen. The effect of each component of thebioadhesive formulation on the release profile from the correspondingbioadhesive matrix was examined through changing its concentration inthe formulation while keeping the concentrations of the other componentsunchanged.

The concentration range for testing was determined according to theactual ability to apply the formulations, from both types of aspects,viscosity and toxicity (especially the toxic coupling agent).

(a) Bupivacaine Release:

In order to examine the gelatin effect on the release profile ofbupivacaine, release profiles of bioadhesive matrices made frombioadhesive formulations with two gelatin concentrations, 100 mg/ml and200 mg/ml, were measured. The obtained data is presented in FIG. 7.

As can be seen in FIG. 7, the effect of gelatin concentration in thepre-curing bioadhesive formulation on the release rate of bupivacaine issubtle; a decrease in the gelatin concentration reduces the burst effectof the drug from the corresponding bioadhesive matrix.

In order to examine the alginate effect on the release profile ofbupivacaine, release profiles from bioadhesive formulations of threedifferent alginate concentrations, 20 mg/ml, 40 mg/ml and 60 mg/ml, werecompared. The obtained data is presented in FIG. 8.

As can be seen in FIG. 8, a decrease in the alginate concentration inthe pre-curing bioadhesive formulation increases the burst effect of thedrug from the corresponding bioadhesive matrix.

In order to examine the EDC effect on the release profile ofbupivacaine, release profiles from bioadhesive matrices prepared frompre-curing bioadhesive formulations containing five different EDCconcentrations, 0 mg/ml, 4 mg/ml, 10 mg/ml, 15 mg/ml and 20 mg/ml, werecompared. The obtained data is presented in FIG. 9.

As can be seen in FIG. 9, an increase in the EDC concentration decreasesthe burst effect of bupivacaine.

In order to examine the effect of drug content in a pre-curingbioadhesive formulation on its release profile from the correspondingbioadhesive matrix, release profiles of bupivacaine from matricesafforded from bioadhesive formulations containing three bupivacaineconcentrations, 1% w/v, 2% w/v and 3% w/v, were compared. The obtaineddata is presented in FIG. 10.

As can be seen in FIG. 10, increasing the bupivacaine concentrationdecreases the burst effect of the drug.

It should be noted that in all matrices prepared from the exemplarytested bioadhesive formulations, approximately 99% of the encapsulatedbupivacaine was released during the first 3 days of the experiment.Also, 100% of the drug was considered the total amount of bioactiveagent that was released until the end of the experiment after 2 weeks,since the drug amount that remained in the matrix after the 2 weeksrelease was found to be negligible, which is reasonable considering thefact that bupivacaine is a hydrophilic drug which is released from thehydrophilic coupled gelatin-alginate matrix. The burst release valuesfrom all studied samples are presented in Table 3.

TABLE 3 Alginate EDC Bupivacaine Gelatin content content content contentBurst effect [mg/ml] [mg/ml] [mg/ml] [% w/v] (six hours) [%] 100 40 20 343.68 ± 0.72 200 51.28 ± 0.90 200 20 20 3 54.33 ± 1.74 40 51.28 ± 0.9060 46.53 ± 1.60 200 40 0 3 100.00 ± 0.00  4 74.36 ± 2.60 10 61.24 ± 1.3615 55.09 ± 1.37 20 51.28 ± 0.90 200 40 20 1 69.67 ± 2.35 2 59.17 ± 2.213 51.28 ± 0.90

The examination of the influence of EDC on bupivacaine releasedemonstrates the role of swelling and water penetration in the drugrelease mechanism. It was noticed that increasing the EDC concentrationdecreases the burst effect of bupivacaine.

In general, in a glassy state of the hydrogel, the diffusion ability ofthe drug is negligible, and when the hydrogel is hydrated sufficiently,it becomes rubbery and the drug can diffuse out.

Support for this result can be found in the water uptake tests whichshowed that the EDC concentration has a significant influence on thewater uptake of the resulting bioadhesive matrix in the first hours ofthe release (see, FIG. 15 and Table 5 hereinbelow). Increasing the EDCconcentration decreases the water uptake of the resulting bioadhesivematrix from the aforementioned reasons.

It can be seen from the release curves that bioadhesive matrices with noEDC had a 100% burst effect release because gelatin and alginate werenot chemically crosslinked to any significant extent and therefore theformulation dissolved completely.

The drug concentration influence on its release rate experiment,demonstrates the diffusion-controlled character of the release. As canbe seen from the release curves, although bioadhesive matrices made fromformulations having a higher concentration of drug actually release agreater mass of drug in the burst release time-range, the burst effectis still smaller relative to the total amount of loaded drug.

The water uptake results (see, Example 5 below) showed that both gelatinand alginate have a similar effect on the swelling of the bioadhesivematrices in the first hours. Decreasing the concentration of bothgelatin and alginate decrease also the water uptake of the correspondingbioadhesive matrix for two possible reasons.

It is noted that the isoelectric points (pKa) of bupivacaine andalginate are 8.1-8.4 and 3.4-4.4, respectively. This means that in therelease conditions of pH 7, the bupivacaine molecules are positivelycharged and the alginate molecules are negatively charged. Thus, anelectrostatic attraction occurs between bupivacaine and alginate.Therefore it is reasonable to assume that the delay in bupivacainerelease when increasing the alginate concentration is as a result ofelectrostatic attraction.

The isoelectric point of gelatin type A, which has both amine andcarboxylic groups in its side chains, is 7.0-9.0, which means that ifnot neutral, gelatin is positively charged in pH 7. Therefore, anelectrostatic repulsion between the gelatin chains and the alsopositively charged bupivacaine molecules is expected, and it can beassumed that this repulsion is increased when the gelatin concentrationin increased. This contributes also to a greater burst effect ofbupivacaine besides water swelling.

The overall examination of the effects of all three components of thebioadhesive formulation (gelatin, alginate and EDC) on the releaseprofile of a given concentration of bupivacaine in the bioadhesivematrix shows that the EDC has a more noticeable effect on thedrug-release profile compared to that of gelatin and alginate.

(b) Ibuprofen Release:

The effect of EDC and drug content in the bioadhesive formulation on therelease kinetics of ibuprofen was tested for bioadhesive matrices,according to some embodiments of the present invention.

Only a fraction of the encapsulated ibuprofen was released in the timeperiod of the experiment. The appearance of two unidentified peaks (onesinglet peak and one doublet peak) in the HPLC chromatogram, indicatedthat a portion of the ibuprofen molecules react with certain componentsin the pre-curing bioadhesive formulation to afford ibuprofenderivatives. As a result, creating ibuprofen release curves requireddefining a calculated theoretical amount of the loaded drug, based onthe weight of the air-dried bioadhesive matrix samples, as the 100%amount of drugs in the samples.

In order to examine the EDC effect on the release profile of ibuprofen,release profiles from three matrices made from bioadhesive formulationshaving three different EDC concentrations were compared, 10 mg/ml, 15mg/ml and 20 mg/ml EDC. The obtained data is presented in FIG. 11.

As can be seen in FIG. 11, increasing the EDC concentration in thebioadhesive formulation decreases the burst effect of ibuprofen from thecorresponding matrix and the total efficiency of released ibuprofen.

In order to examine the ibuprofen effect on its release profile, threedifferent ibuprofen concentrations were used in preparing bioadhesiveformulations, 1% w/v, 2% w/v and 3% w/v ibuprofen. The obtained data ispresented in FIG. 12.

As can be seen in FIG. 12, increasing the ibuprofen content in thebioadhesive formulation increases the efficiency of the total pristineibuprofen that is released from the corresponding matrix, while theburst effect is affected marginally by the ibuprofen concentration.

The burst effect (first six hours) and total pure ibuprofen releasedfrom all studied samples are presented in Table 4.

TABLE 4 Cumulative Gelatin Alginate EDC Ibuprofen ibuprofen contentcontent content content Burst effect released [mg/ml] [mg/ml] [mg/ml] [%w/v] (six hours) [%] [%] 200 40 10 3 47.85 ± 2.23 68.09 ± 1.19 15 35.59± 1.13 56.10 ± 0.75 20 29.85 ± 1.95 52.84 ± 1.33 200 40 20 1 26.93 ±1.12 36.30 ± 2.23 2 28.26 ± 0.77 44.18 ± 0.67 3 29.89 ± 2.73 52.04 ±2.13

As mentioned above, in view of the results it has been assumed that theibuprofen molecule reacts with certain components in the bioadhesiveformulation to afford ibuprofen derivatives. The reason for theibuprofen's reactivity in the bioadhesive formulation is attributed toits carboxylic group.

The observed results of the release curves of ibuprofen support theassumed reaction between ibuprofen and EDC. Increasing the EDCconcentration in the bioadhesive formulation decreased the burst effectof ibuprofen from the corresponding matrix, as expected, but also theefficiency of pristine ibuprofen release. As the EDC concentration inthe formulation is increased under a constant concentration of the drug,the relative fraction of ibuprofen that reacts with the EDC grows aswell. Similarly, increasing the ibuprofen concentration in thebioadhesive formulation increases its release efficiency, since at aconstant concentration of EDC the relative fraction of ibuprofen thatreacts with the EDC decreases.

Example 5 Initial Water Uptake

The effects of the concentration of various components in thebioadhesive formulations, according to some embodiments of the presentinvention, on the initial water uptake of the resulting matrices duringthe first six hours from preparation were examined. As a referenceformulation a bioadhesive formulation containing 200 mg/ml gelatin, 40mg/ml alginate and 20 mg/ml EDC was chosen.

Similarly to the drug release tests, the concentration range for theinitial water uptake testing was determined according to the actualability to apply the formulations in terms of viscosity in the case ofgelatin and alginate, or toxicity in the case of EDC.

According to these criteria, the effect of gelatin concentration wasexamined on formulations containing 100 mg/ml and 200 mg/ml gelatin, theeffect of alginate concentration was examined on formulations containing20 mg/ml and 40 mg/ml alginate and the effect of EDC concentrations wasexamined on formulations containing 10 mg/ml and 20 mg/ml EDC, and theresults are presented in FIG. 13, FIG. 14 and FIG. 15, respectively.

As can be seen in FIG. 13, FIG. 14 and FIG. 15, decrease in the EDCconcentration increased the water uptake by the bioadhesive matrices,while decrease in the gelatin or alginate concentrations in theformulations decreased the water uptake by the corresponding bioadhesivematrix.

Table 5 summarizes the initial water uptake measured for exemplarybioadhesive matrices during the first six hours.

TABLE 5 Gelatin Alginate EDC content content content Initial wateruptake [mg/ml] [mg/ml] [mg/ml] (six hours) [%] 100 40 20 200.14 ± 2.72 200 275.58 ± 16.69 200 20 20 244.94 ± 10.44 40 275.58 ± 16.69 200 40 10528.10 ± 44.60 20 275.58 ± 16.69

Example 6 Cytotoxicity

In one experiment the effect of each component of the bioadhesiveformulation, according to some embodiments of the present invention, oncytotoxicity was tested.

The obtained data is presented in FIG. 16.

As can be seen in FIG. 16, the results after 24 hours, compared to thecontrol, show that the alginate or gelatin are biocompatible and do notinduce any cytotoxic effects.

In another experiment, the effect of exemplary bioadhesiveformulation/matrix on cells' viability was demonstrated by exposingcells to formulations based on 200 or 300 mg/ml gelatin and 40 or 30mg/ml alginate and varying EDC concentrations (0, 5, 10, 15 and 20mg/ml), and the results are presented in FIGS. 17A-B.

As can be seen in FIGS. 17A-B, as the concentration of EDC increases,the cell viability decreases. When cells were exposed to bioadhesivematrices based on 200 mg/ml gelatin and 40 mg/ml alginate and lowconcentrations of EDC (0, 5 and 10 mg/ml), the cells exhibit relativelyhigh viability (89-100%), while at higher EDC concentrations (15 and 20mg/ml), the cell viability decreases to 73% and 71%, respectively. Thistendency was observed also after 48 hours of incubation with the mediumthat contains the bioadhesive matrices. When cells were exposed tobioadhesive formulations based on 300 mg/ml gelatin and 30 mg/mlalginate, low concentrations of the EDC (0, 5 and 10 mg/ml) results inrelatively high cell viability (88-96%) while at higher EDCconcentration (15 and 20 mg/ml) the cell viability decreases to 76% and71%, respectively. In general, it can be concluded that relatively highviability is maintained even at relatively high EDC concentrations.

Therefore it can be concluded that the bioadhesive matrices affordedfrom bioadhesive formulations according to some embodiments of thepresent invention, are biocompatible and can safely be used.

Fibroblast viability was also examined after exposure to aqueoussolutions of 1, 2 and 3% w/v bupivacaine and ibuprofen for 24 hours, bymeasuring their Alamar Blue reduction relative to the Alamar Bluereduction of control cells. The obtained data is presented in FIGS.18A-B.

As can be seen in FIGS. 18A-B, some cytotoxic effects were obtained forboth bupivacaine and ibuprofen when testing concentrations higher than1% w/v. The cell viability values in the presence of bupivacaine (67.6%and 58.8% for 2% w/v and 3% w/v drug, respectively) were lower thanthose obtained in the presence of ibuprofen (93.5% and 79.7% for 2% w/vand 3% w/v drug, respectively). It is noted herein that at least 50%cell viability was achieved in all cases, compared to the control. It isalso noted herein that this test is characterized by a tendency toafford low cell viability results compared to the experiments usingdrug-loaded bioadhesive matrices since the drugs are released graduallyfrom bioadhesive matrix over 3 days, which is different from theconditions in this experiment.

Example 7 Microstructure Characterization

The bulk cross-sections of air-dried bioadhesive matrix specimens,according to some embodiments of the present invention, prepared withconcentrations of gelatin (200 mg/ml), alginate (40 mg/ml) and EDC (20mg/ml) and pre-loaded with 1% w/v bupivacaine or ibuprofen, wereobserved using environmental scanning electron microscope (ESEM). Theobtained data is presented in FIG. 19.

As can be seen in FIGS. 19A-D, the drug-free reference sample did notdemonstrate any phase separation, and exhibited some cracking whichprobably resulted from the fracturing process, while the fractographs ofbupivacaine-loaded bioadhesive matrices provide a clear perspective ofthe dispersion and crystallization of the drug within the bioadhesivematrix. As can be seen in FIGS. 19B-D, bupivacaine is uniformlydispersed within the matrix and crystallizes into two levels ofstructure: a primary structure of needles that form fiber-shapedsecondary structures. This type of crystallization apparently turns thebupivacaine-loaded bioadhesive matrix into a kind of fiber-reinforcedcomposite material.

Evaluation of the fibers' diameter was performed by measuring thediameters of 50 fibers from the same area for 3 different specimens(total of 150 fibers). Evaluation of the needles' diameter was performedby measuring the diameters of 40 needles per fiber, for 3 differentfibers (total of 120 needles). The bupivacaine-loaded bioadhesive matrixexhibited mean fiber and needle diameters of 11.69±2.49 and 0.49±0.09μm, respectively. Regardless of the size of the bupivacaine crystals,their homogenous dispersion in the matrix indicates that thebupivacaine-loaded bioadhesive matrix is actually a type of monolithicsystem, exhibiting release profiles which are typical for monolithicdevices.

The structure of the ibuprofen-loaded bioadhesive matrix samples hasalso been characterized. The presence of ibuprofen in the bioadhesivematrix samples could be detected in certain domains. An example for sucha domain is shown in FIGS. 20A-B.

As can be seen in FIGS. 20A-B, the ibuprofen crystals were randomlydistributed in the bioadhesive matrix in needle-shaped structureswithout any secondary structure.

Without being bound to any particular theory, it is assumed thatibuprofen, similarly to bupivacaine, is also uniformly dispersed in thematrix since it was mixed with the bioadhesive formulation components inthe same way, and because its release profiles exhibited the samebehavior of a decrease with time which is typical for monolithicsystems.

Example 8 Bioadhesive Formulation Containing Fillers

The preparation of bioadhesive formulations containing the exemplaryfillers, hydroxyapatite (HA) and beta-tricalcium phosphate (β-TCP), isbased on the procedure for preparing bioadhesive formulation without afiller, and is carried out by dissolving various amounts of gelatin,alginate and filler powders in distilled water while heating to 60° C.Various amounts of a crosslinking agent (EDC) are added immediatelyprior to the bioadhesive's use. All studied formulations containingfillers are presented in Tables 6 and Table 7 below.

Table 6 presents the studied bioadhesive formulation containing HA andβ-TCP for soft tissue adhesion.

TABLE 6 Filler Gelatin Alginate EDC concen- concentration concentrationconcentration tration Filler (mg/ml) (mg/ml) (mg/ml) (% w/v) Filler-free200 40 20 0 HA 200 40 20 0.125 0.25 0.5 10 0.125 0.25 0.5 β-TCP 200 4020 0.125 0.25 0.5 10 0.125 0.25 0.5

Table 7 presents the studied bioadhesive formulation containing HA andβ-TCP for hard tissue adhesion.

TABLE 7 Gelatin Alginate EDC Filler Filler content concentrationconcentration concentration concentration in dry samples Filler (mg/ml)(mg/ml) (mg/ml) (% w/v) (% w/w) Filler-free 200 40 20 0 0 HA 200 40 200.25 1 β-TCP 200 40 20 0.5 2

Example 9 Filler Effect on Soft Tissue Bonding Strength

The bonding strength of HA-loaded and β-TCP-loaded bioadhesiveformulations to soft tissues, was measured at various fillerconcentrations: 0.125%, 0.25% and 0.5% w/v. These relatively lowconcentrations were chosen since while HA and β-TCP are essentiallyinsoluble in aqueous solutions, these fillers could nonetheless beloaded inside the bioadhesive hydrogel with only minimal precipitation.The concentrations of gelatin (200 mg/ml), alginate (40 mg/ml) and EDC(20 mg/ml) were used in this study.

The bonding strength results were compared to the bonding strength ofbioadhesive formulations prepared without fillers. Fifteen repetitionswere carried out for each formulation, and the results are presented inFIG. 21.

As can be seen in FIG. 21A, incorporation of both HA and β-TCP increasedthe bonding strength of the bioadhesive matrices. An increase of thebonding strength with HA was afforded at concentrations of 0.25% w/v andhigher. The highest bonding strength was observed for a HA concentrationof 0.25% w/v, 18.1±4.0 kPa compared to 8.4±2.3 kPa for the correspondingbioadhesive without a filler. Increasing the HA concentration to 0.5%w/v lowered the bonding strength (13.4±1.6 kPa), however, it was stillhigher than the bonding strength of the reference formulation. As canfurther be seen in FIG. 21B, all examined β-TCP concentrations werefound to increase the bonding strength, with no significant differenceobserved between the samples prepared with different concentrationsthereof. The highest average value was measured for a β-TCPconcentration of 0.5% w/v (15.2±2.6 kPa).

A relatively low concentration of a crosslinking agent is beneficial interms of biocompatibility and reduced cytotoxicity. Hence, the effect offillers on the bonding strength of bioadhesive formulations with areduced concentration of EDC (10 mg/ml) was studied as well using thesame three concentrations of the fillers, namely 0.125%, 0.25% and 0.5%w/v) and the same for the polymeric components gelatin (200 mg/ml) andalginate (40 mg/ml) concentrations. Fifteen repetitions were carried outfor each formulation, and the results are presented in FIGS. 22A-B.

As can be seen in FIG. 22A, incorporation of HA-loaded and β-TCP-loadedbioadhesive formulations prepared with reduced amount of EDC (10 mg/ml)had bonding strengths similar to the bonding strength of the filler-freebioadhesive with EDC concentration of 20 mg/ml. Bioadhesive matricescontaining reduced EDC (10 mg/ml) and concentrations of 0.5% and 0.125%w/v HA or β-TCP respectively, were found to have even higher bondingstrength.

The bonding strength of Evicel™, a commercial fibrin glue, was alsomeasured using the same bonding strength system, and a comparisonbetween the bonding strength of Evicel™ (2.5±2.3 kPa) and a bioadhesivematrix containing HA and β-TCP, according to some embodiments of thepresent invention, showed that even when the EDC concentration wasreduced by half, incorporation of the fillers enable the achievement ofup to 7 times higher bonding strength to soft tissues compared withEvicel™

Example 10 Filler Effect on Hard Tissue Bonding Strength

HA-loaded and β-TCP-loaded bioadhesive matrices, according to someembodiments of the present invention, were also examined for theirpotential use in hard tissue adhesion.

0.25% w/v HA and 0.5% w/v β-TCP were added to exemplary bioadhesiveformulations, according to some embodiments of the present invention,and the bonding strength results were compared to a filler-freereference bioadhesive formulation, with the concentrations of thepolymeric components maintained the same for all tests (EDCconcentration set to 20 mg/ml). Three repetitions were carried out foreach formulation, and the results are presented in FIG. 23.

As can be seen in FIG. 23, adding 0.25% w/v HA almost tripled thebonding strength of the bioadhesive matrix from 26.6±9.2 kPa to71.4±28.2 kPa, while the sample containing β-TCP did not exhibit thesame effect.

The adherence ability of other bioadhesives to hard tissues wasevaluated and it was found that under similar test conditions, fibrinand gelatin-resorcinol-formaldehyde formulations exhibit ex vivo bondingstrengths of 11 and 200 kPa, respectively. The bioadhesiveformulations/matrices according to embodiments of the present invention,which incorporated HA particles exhibit bonding strength values to hardtissues that is 6.5-times higher than that of fibrin (71.4±28.2 kPa),and lower than that of the gelatin-resorcin-formaldehyde adhesive, whichis considered to be less biocompatible than the bioadhesive formulationpresented herein (formaldehyde being significantly more cytotoxic thanEDC).

Example 11 Filler Effect on Microstructure of the Matrix

The bulk cross-sections of air-dried bioadhesive matrix specimenscontaining 0.125% and 0.5% w/v HA and β-TCP respectively were examinedusing ESEM. The concentrations of gelatin (200 mg/ml), alginate (40mg/ml) and EDC (20 mg/ml) were kept constant for all samples. Themicrostructure analysis of a filler-free bioadhesive presentedhereinabove has shown that no phase separation occurred in the presenceof the three basic components of the bioadhesive formulation, gelatin,alginate and EDC.

The fractographs of the HA-loaded and β-TCP-loaded bioadhesiveformulations are presented in FIGS. 24A-F.

As can be seen in FIGS. 24A-F, the fractographs provide a clearperspective on the crystallization of the fillers in the bioadhesivematrix. The fractographs also demonstrate that at the lowerconcentration, the HA and β-TCP particles are not uniformly dispersed inthe adhesive. Higher concentrations of the fillers seem to provide amore uniform dispersion.

The soft tissue bonding strength experiments demonstrated that both HAand β-TCP can be regarded as effective reinforcement fillers, sinceadding these fillers to the bioadhesive formulation increases itsbonding strength.

The crosslinking agent concentration had a significant effect on thebonding strength since it controls the crosslinking density in thebioadhesive matrix. As presented hereinabove, decreasing thecrosslinking agent concentration by half, from 20 mg/ml to 10 mg/ml,caused about two-fold reduction in the bonding strength of a bioadhesivematrix stemming from the same formulation. Comparing the bondingstrength results of HA-loaded and β-TCP-loaded bioadhesive matrices witha reduced concentration of EDC (10 mg/ml) to that of the filler-freereference bioadhesive (20 mg/ml EDC) indicated that both HA and β-TCPhave a compensatory effect on the bonding strength when decreasing thecrosslinking agent concentration. Since EDC was shown to have somecytotoxic effects, this compensatory effect also has clinical andmedical importance because it enables improving the biocompatibility ofthe adhesive without compromising its bonding strength and evenimproving it.

Example 12 Adhesion Mechanism

The two main adhesion mechanisms were tested using the bioadhesiveformulations/matrices presented herein:

(1) Mechanical interlocking—adhesion as a result of penetration of theadhesive into pores, grooves and other irregularities on the surface ofthe adherents.

(2) Chemical adhesion—adhesion as a result of intramolecular bonds (vander Waals, ionic, covalent, hydrogen and/or metallic) between theadhesive agent and molecules on the surface of the adherents.

For most adhesives, the adhesion mechanism is considered to be acombination of mechanical interlocking and chemical adsorption. Theproportionate contribution of each of the two mechanisms to the finalbonding strength is variable and affected by the adhesive type, thesurface roughness and the environment.

In an attempt to elucidate the adhesive mechanism of the bioadhesiveformulation/matrix presented herein, the effects of gelatin, alginateand the crosslinking agent's concentrations and their viscosities on theability of the bioadhesive to bind to soft tissues was furtherinvestigated. Accordingly, a qualitative model describing these effectsin terms of adherence mechanisms is presented hereinbelow.

All studied formulation series are presented in Tables 8-10.

Table 8 presents the studied bioadhesive formulations, according to someembodiments of the present invention, with different EDC concentrationsand various gelatin-alginate combinations, whereas “LV” denotes lowviscosity (0.1-0.3 Pa-sec).

TABLE 8 Gelatin (90-110 Bloom, mg/ml) LV Alginate (mg/ml) EDC (mg/ml)200 40 5 10 15 20 300 30 5 10 15 20

Table 9 presents the studied bioadhesive formulations with differentgelatin Bloom numbers, whereas “LV” denotes low viscosity (0.1-0.3Pa-sec).

TABLE 9 Gelatin EDC concentration Gelatin Bloom LV Alginateconcentration (mg/ml) number concentration (mg/ml) (mg/ml) 200 90-110 4020 175 300

Table 10 presents the studied bioadhesive formulations with differentalginate concentrations and viscosities under various gelatinconcentrations, whereas “LV” denotes low viscosity (0.1-0.3 Pa-sec) and“HV” denotes high viscosity (more than 2 Pa-sec).

TABLE 10 Gelatin (90-110 Bloom Alginate EDC number) concentrationconcentration Alginate concentration (mg/ml) (mg/ml) viscosity* (mg/ml)200 10 LV 20 HV 20 LV HV 30 LV HV 40 LV 300 10 LV 20 30 400 10 20 30

The ex vivo bonding strength measurements were conducted as describedhereinabove for the soft tissue studies.

Effect of the EDC Concentration:

The effect of EDC on the bonding strength was evaluated by measuring thebonding strengths of two main combinations, 200 mg/ml gelatin with 40mg/ml alginate (Ge-200:Al-40), and 300 mg/ml gelatin with 30 mg/mlalginate (Ge-300:Al-30), with a gelatin Bloom number of 90-110 and LValginate, and with 5, 10, 15 and 20 mg/ml EDC concentrations (see, Table8) and the results are presented in FIGS. 25A-B.

As can be seen in FIGS. 25A-B, increasing the EDC concentration resultsin a significant increase in the bonding strength of bothgelatin-alginate formulations. For the Ge-200:Al-40 formulation,increasing the EDC concentration from 5 to 20 mg/ml increased thebonding strength of the bioadhesive from 3.2±0.8 to 11.5±2.0 kPa (FIG.25A), and for the Ge-300:Al-30 formulation, increasing the EDCconcentration from 5 to 20 mg/ml increased the bonding strength of thebioadhesive from 5.1±1.1 to 13.0±2.7 kPa (FIG. 25B).

Increasing the EDC concentration leads to a denser bioadhesivecrosslinked network. A denser network results in better mechanicalproperties and greater cohesion strength of the adhesive, whichcontribute to the mechanical interlocking mechanism of adhesion. Ahigher crosslinking density can also result in a higher density ofcovalent bonds between exposed functional groups in the lacerated tissueand the adhesive, which contribute to adhesion through the chemicalabsorption mechanism.

Effect of the Gelatin Bloom Number:

The Bloom number can give an indication for the strength of the gelformed from a solution with a known concentration. Stronger gels arecharacterized by higher Bloom numbers. The Bloom number also reflectsthe average molecular weight of its constituents. The effect of thegelatin Bloom number on the bonding strength was examined by measuringthe bonding strength of Ge-200:Al(LV)-40:EDC-20 bioadhesives with threedifferent gelatin Bloom numbers, 90-110, 175 and 300. The viscosity ofthe non-crosslinked Ge—Al solution of each of these bioadhesiveformulations was also evaluated (see, Table 9). The results arepresented in FIGS. 26A-B.

As can be seen in FIG. 26A, increasing the gelatin Bloom number resultedin a consistent and significant decrease in the bonding strength of thebioadhesive, from 11.5±2.0 kPa for gelatin with a Bloom number of 90-110to 6.4±1.6 kPa for gelatin with a Bloom number of 300. The increase inthe molecular weight of gelatin, i.e. increase in the Bloom number,decreases the bioadhesive's mobility and probably also decreases thegelatin strings' ability to penetrate into pores or irregularities onthe surface of the soft tissue. The contribution of mechanicalinterlocking to the general adherence effect of the adhesive to thetissue is therefore reduced, and the bonding strength decreases.

Support for the decrease in the bioadhesive's mobility when increasingthe gelatin Bloom number can be found in the initial viscosity results.As can be seen in FIG. 26B, increasing the gelatin Bloom number from90-110 to 300 resulted in a significant increase in the viscosity, from12.5 to 38.8 Pa-sec.

Effect of the Polymeric Component Concentrations:

The effect of both polymeric component concentrations on the bondingstrength of the bioadhesive was examined using gelatin with a Bloomnumber of 90-110 based on the results presented hereinabove. Bioadhesiveformulations with gelatin concentrations of 200, 300 and 400 mg/ml andLV alginate concentrations of 10, 20, 30 and 40 mg/ml were examined witha constant EDC concentration of 20 mg/ml (see, Table 10). It is notedherein that for bioadhesive formulations based on a gelatinconcentration higher than 200 mg/ml, 30 mg/ml was the highest alginateconcentration that was tested, since higher alginate concentrationsresulted in solutions that were too viscous to be mixed homogenously.The results are presented in FIGS. 27A-C.

As can be seen in FIGS. 27A-C, when a gelatin concentration of 200 mg/mlwas used, maximal bonding strength was achieved for both the highest andthe lowest alginate concentrations (10.3±1.3 and 11.5±2.0 kPa for 10 and40 mg/ml alginate, respectively).

As can further be seen in FIGS. 27B-C, a different effect of thealginate concentration on the bonding strength was obtained forbioadhesive formulations with relatively high gelatin concentrations of300 and 400 mg/ml. A maximal bonding strength of 13.0±2.7 kPa wasmeasured for the highest alginate concentration that was tested, 30mg/ml, in bioadhesive formulations with a gelatin concentration of 300mg/ml (FIG. 27B). The alginate concentration was found to have a smalleffect on the bonding strength in bioadhesive formulations with agelatin concentration of 400 mg/ml (FIG. 27C). This different behaviorprobably indicates that when the gelatin concentration is relativelyhigh, the alginate concentration (which is significantly less than thegelatin concentration) has a minor effect on the crosslinking densityand the entanglement level of the 3-dimensional structure of theadhesive, and therefore exhibits only a small effect on the bondingstrength.

Effect of Alginate's Viscosity:

The alginate's viscosity correlates to the average molecular weight ofits chains. Formulations based on gelatin concentrations of 200, 300 and400 mg/ml and alginate concentrations of 10, 20 and 30 mg/ml were used.Each formulation was tested with both low viscosity (LV alginate) andhigh viscosity (HV) alginate, and the EDC concentration was kept at 20mg/ml. It is noted herein that solutions of 30 mg/ml HV alginate weretoo viscous to be mixed homogenously with gelatin concentrations higherthan 200 mg/ml, and a bioadhesive solution containing 40 mg/ml HValginate could not be mixed homogenously even with only 200 mg/mlgelatin. The examined formulations are presented in Table 10 and theresults are presented in FIGS. 28A-C.

As can be seen in FIGS. 28A-C, HV alginate bioadhesive formulationsbased on 200 mg/ml gelatin were found to exhibit a similar effect ofconcentration on the bonding strength as LV alginate, i.e., the highestbonding strengths were obtained for the highest and lowest HV alginateconcentrations used in this study (9.8±2.3 and 8.1±1.4 kPa for 10 and 30mg/ml HV alginate, respectively; FIG. 28A). The effect of alginate'sviscosity on the bonding strength was found to be opposite to that ofgelatin's viscosity (Bloom number). Use of HV alginate instead of LValginate slightly improved the bonding strength of the adhesive, exceptfor the lowest alginate concentration that was examined (10 mg/ml),where no significant difference was observed.

The effect of alginate's viscosity on the total viscosity of thegelatin-alginate solution for formulations based on 200 mg/ml gelatin(90-110 Bloom number) with various alginate concentrations is presentedin FIG. 29.

As can be seen in FIG. 29, the viscosity of the bioadhesive formulationprecursor (gelatin-alginate solution) is increased 5-6 times when HValginate is used instead of LV alginate. However, this change enablesonly a minor improvement in the bonding strength of the bioadhesivematrix resulting from a formulation of similar gelatin-alginate content(see, FIG. 28A). This phenomenon indicates that although alginate'sviscosity strongly affects the viscosity of the gelatin-alginatesolution, it has only a minor effect on the bioadhesive's strength (FIG.28A). This may mean that it is possible to create bioadhesiveformulations with similar adherence qualities but different viscositiesthat may be tailored for different applications.

A Formulation—Strength Model:

A qualitative model which describes the effect of the bioadhesiveformulation's parameters on the bonding strength of the resultingbioadhesive matrix is suggested based on the findings presentedhereinabove. A schematic representation of this model is presented inFIG. 30.

Example 13 Combined Effect of EDC and NHS

N-hydroxysuccinimide (NHS) was added to the crosslinking reaction ofgelatin with EDC in order to improve the crosslinking reactionefficiency, lower the side reactions and lower the amounts of EDC thatare needed effect effective crosslinking in the bioadhesive formulationsaccording to some embodiments of the present invention.

This study was designed to identify the optimal amounts of EDC and NHSthat affords an effective bioadhesive formulation based on gelatin andalginate. NHS was tested at contents of 0, 10, 20, 40 and 50% (inpercent of the amount of EDC) and the results are presented in FIG. 31.

As can be seen in FIGS. 31A-B, incorporation of NHS in the bioadhesiveformulation presented herein affords bioadhesive matrices which exhibithigher bonding strengths while using less of crosslinking agent EDC.

These experiments demonstrate that EDC concentrations can be loweredsignificantly with the addition of relatively small amounts of othercrosslinking agents such as NHS.

Example 14 Bioadhesive Formulations Loaded with Antibiotic Drugs

Clindamycin is well known for its activity against Gram-positive cocci,Gram-positive and Gram-negative anaerobes and certain protozoa. Despitethe favorable antimicrobial spectrum of clindamycin, its systemicadministration is associated with a serious antibiotic-relatedcomplication named pseudomembranous enterocolitis. Therefore, deliveringthis drug locally could potentially decrease the risk of systemiccomplications. Hence, the study presented herein is aimed atinvestigating the possibility of loading the bioadhesiveformulations/matrices with antibiotic drugs such as clindamycin.

Bioadhisive formulations containing an antibiotic drug were prepared asdescribed hereinabove, and the mechanical properties of the resultingbioadhesive matrices were measured as described hereinabove. The resultsof the bonding strength measurements of exemplary clindamycin-loadedbioadhesive formulations according to some embodiments of the presentinvention, are presented in FIGS. 32A-B.

As can be seen in FIGS. 32A-B, since clindamycin does not exhibitcarboxylic or primary amines groups, and therefore it is inert to thecrosslinking reaction, its presence did not decrease the bondingstrength but rather increased it to some extent.

The release profile of clindamicin was also measured according to theprotocols presented hereinabove. These studies showed that most of theantibiotic drug was released from the bioadhesive matrix within 6 hoursin all studied formulations containing various concentrations of thedrug.

Example 15 Bioadhesive Formulations Loaded with Haemostatic Agents

Incorporation of haemostatic agents in the bioadhesive formulationspresented herein may be beneficial for various medicinal applications.Hence, the bonding strength of exemplary bioadhesive matrices, formedfrom bioadhesive formulations prepared with the exemplary haemostaticagents kaolin and tranexamic acid, was investigated, and the results arepresented in Table 11.

The exemplary bioadhesive formulations which were chosen for this studyand the average bonding strength of the resulting bioadhesive matrix arepresented in Table 11.

TABLE 11 Bonding Gelatin Alginate EDC NHS strength Formulation (mg/ml)(mg/ml) (mg/ml) (% of EDC) (kPa) 0 200 40 20 0 13.3 1 200 40 10 10 13.32 300 30 10 10 17.4 3 300 30 20 0 18.4

The effect of incorporation of the exemplary haemostatic agents kaolinand tranexamic acid in the bioadhesive formulations are presented inTable 12.

TABLE 12 Formulation 0 1 3 5 7 Kaolin content in % w/v 1 13.3 17.5 25.519.5 15.5 2 17.4 19.6 19 16.6 17.7 3 18.4 16.7 20.9 22.4 20.6 Tranexamicacid content in % w/v 1 13.3 9.8 — 2 17.4 13.9 8.4 3 18.4 13.4 10.5

As can be seen, incorporation of kaolin increases the bonding strengthof two of the studied formulations, while incorporation of tranexamicacid resulted in a decreased bonding strength.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention. To the extent thatsection headings are used, they should not be construed as necessarilylimiting.

What is claimed is:
 1. A kit for preparing a bioadhesive formulation, comprising sub-formulation A and sub-formulation B, said sub-formulation A comprises gelatin and alginate dissolved in water and said sub-formulation B comprises a carbodiimide, whereas combining said sub-formulation A with said sub-formulation B affords the bioadhesive formulation, the bioadhesive formulation being for forming a bioadhesive matrix upon allowing a curing time to elapse, and wherein upon said combining: a concentration of said gelatin in the bioadhesive formulation ranges from 50 mg/ml to 400 mg/ml, a concentration of said alginate in the bioadhesive formulation ranges from 10 mg/ml to 60 mg/ml, and a concentration of said carbodiimide in the bioadhesive formulation ranges from 5 mg/ml to 50 mg/ml, such that prior to curing, the bioadhesive formulation is characterized by a room temperature viscosity that ranges from 1 Pa-sec to 50 Pa-sec, and such that said curing time ranges from 5 seconds to 30 minutes.
 2. The kit of claim 1, wherein said carbodiimide in said sub-formulation B is dissolved in water.
 3. The kit of claim 1, wherein said matrix is characterized by at least one of: a bonding strength of viable biological objects that ranges from 2,000 pascal to 60,000 pascal; a flexural strength at physiological conditions that ranges from 0.5 MPa to 200 MPa; and a biodegradability rate that ranges from 7 days to 6 months.
 4. The kit of claim 1, wherein the concentration of said gelatin in the formulation ranges from 200 mg/ml to 300 mg/ml, the concentration of said alginate in the formulation ranges from 20 mg/ml to 40 mg/ml and the concentration of said carbodiimide in the formulation ranges from 10 mg/ml to 30 mg/ml.
 5. The kit of claim 1, wherein said sub-formulation A and/or said sub-formulation B further comprises a crosslinking promoting agent.
 6. The kit of claim 5, wherein a concentration of said crosslinking promoting agent in said formulation ranges from 1% to 50% relative to the amount of said carbodiimide in said formulation.
 7. The kit of claim 1, wherein said sub-formulation A and/or said sub-formulation B further comprises a filler.
 8. The kit of claim 7, wherein a concentration of said filler in said formulation ranges from 0.1% w/v to 1% w/v of the formulation.
 9. The kit of claim 1, wherein said sub-formulation A and/or said sub-formulation B further comprises a bioactive agent.
 10. The kit of claim 9, wherein a concentration of said bioactive agent in said formulation ranges from 0.1 percent weight per volume to 10 percent weight per volume of the total volume of said formulation.
 11. The kit of claim 1, being in a form of a dual chamber dispenser, wherein said dispenser comprises at least a chamber A for containing and dispensing said sub-formulation A, and a chamber B for containing and dispensing said sub-formulation B.
 12. A dual chamber dispenser for use in applying the bioadhesive formulation of claim 1, comprising a dual barrel cartridge assembly having a joint delivery port, a mount for coupling with a mixing tube and a mixing tube, wherein a barrel cartridge A of said dual barrel cartridge assembly is configured for dispensing said sub-formulation A, and a barrel cartridge B of said dual barrel cartridge assembly is configured for dispensing said sub-formulation B, such that upon said dispensing, said sub-formulation A and said sub-formulation B are combined to thereby form the bioadhesive formulation. 